Researcher Profile and Settings

Affiliation

• Faculty of Environmental Earth Science　Environmental Biology　Environmental Molecular Biology

Job Title

• Professor

Degree

• Doctor of Engineering (Osaka University)（1994/10 Osaka University）
• Honorary Doctorate (Bioscience, Kasetsart University)（2022/10 Kasetsart University）

Contact information

• morikawaees.hokudai.ac.jp

URL

https://noah.ees.hokudai.ac.jp/emb/morikawalab/

J-Global ID

• 200901006119139020

Research Interests

• Environmental Biotechnology   極限生命工学   環境分子微生物学   Extremity Life Science   Envionmental Molecular Microbiology   

Research Areas

• Environmental science/Agricultural science / Social-ecological systems / Plant Growth-Promoting Bacteria
• Life sciences / Applied microbiology

Educational Organization

•  Bachelor's degree program　School of Science
•  Master's degree program　Graduate School of Environmental Science
•  Doctoral (PhD) degree program　Graduate School of Environmental Science

Academic & Professional Experience

• 2023/04 - Today International Center for Biotechnology, Osaka University Visiting Professor
• 2004/05 - Today 北海道大学教授 大学院地球環境科学研究院 環境生物科学部門
• 2004/05 - Today Professor,Faculty of Environmental Earth Science, Hokkaido University
• 2020/08 - 2026/09 JST/JICA-SATREPS Project Leader
• 2020/08 - 2026/09 Science and Technology Research Partnership for Sustainable Development Project Leader
• 2011/10 - 2020/03 JST-ALCA Project Leader
• 2011/10 - 2020/03 JST-ALCA (Advanced LowCarbon Research and Development Program Project Leader
• 2004/05 - 2006/03 北海道大学教授 大学院地球環境科学研究科 生態環境科学専攻
• 1995/07 - 2004/04 大阪大学助教授 大学院工学研究科 物質・生命工学専攻
• 1995/07 - 2004/04 Associate Professor,Graduate School of Engineering, Osaka University
• 2001/05 - 2002/03 Visiting Researcher, Harvard Medical School
• 2001 - 2002 Ministry of Education,Culture,Sports,Science and Technology
• 1996 - 2001 生物系特定産業技術研究推進機構プログラム 総括研究代表者
• 1996 - 2001 Project Leader,BRAIN
• 1990/04 - 1995/06 大阪大学助手 工学部発酵工学科
• 1990/04 - 1995/06 Research Associate, Osaka University
• 1985/04 - 1989/03 日清製油株式会社研究所 研究員
• 1985/04 - 1989/03 Researcher,The Nisshin Oil Mills Co. Ltd.
• 1986/04 - 1988/03 工業技術院化学技術研究所(出向)
• 1986/04 - 1988/03 Visiting Researcher,MITI

Education

• 1983/04 - 1985/03  Osaka University  Graduate School of Engineering
• 1983/04 - 1985/03  Osaka University  Department of Fermentation Technology, Graduate School of Engineering
• 1979/04 - 1983/03  Osaka University  School of Engineering
• 1979/04 - 1983/03  Osaka University  Department of Fermentation Technology, Faculty of Engineering
• 1976/04 - 1979/03  府立生野高等学校

Association Memberships

• 環境バイオテクノロジー学会   日本生化学会   新資源生物変換研究会 常任幹事   米国微生物学会   日本農芸化学会   日本生物工学会   ポリリン酸研究会   NEDO技術委員   American Society for Microbiology   Study Group of Polyphosphate   Japan Society for Environmental Biotechnology   and Agrochemistry   Biotechnology   Japan Society for Bioscience   Japan Society for Biotechnology   

Research Activities

Published Papers

• A novel alkane monooxygenase evolved from a broken piece of ribonucleotide reductase in Geobacillus kaustophilus HTA426 isolated from Mariana Trench.

  Tanasap Nithimethachoke, Chanita Boonmak, Masaaki Morikawa

  Extremophiles : life under extreme conditions 28 (1) 18 - 18 2024/02/14 [Refereed]
   

  We have accidentally found that a thermophilic Geobacillus kaustophilus HTA426 is capable of degrading alkanes although it has no alkane oxygenating enzyme genes. Our experimental results revealed that a putative ribonucleotide reductase small subunit GkR2loxI (GK2771) gene encodes a novel heterodinuclear Mn-Fe alkane monooxygenase/hydroxylase. GkR2loxI protein can perform two-electron oxidations similar to homonuclear diiron bacterial multicomponent soluble methane monooxygenases. This finding not only answers a long-standing question about the substrate of the R2lox protein clade, but also expands our understanding of the vast diversity and new evolutionary lineage of the bacterial alkane monooxygenase/hydroxylase family.
• Gene cloning and characterization of a vanadium-dependent bromoperoxidase from the red alga Laurencia saitoi, a producer of brominated diterpenoids and triterpenoids

  Kensuke Kaneko, Daiki Kobayashi, Shiro Masaki, Kenji Washio, Masaaki Morikawa, Tatsufumi Okino

  JOURNAL OF APPLIED PHYCOLOGY 0921-8971 2023/04 

  The gene encoding vanadium-dependent bromoperoxidase (V-BPO) was cloned for the first time from the red alga Laurencia saitoi, which produces pharmaceutically promising brominated diterpenoids and triterpenoids. The molecular weight of V-BPO from L. saitoi (LsVBPO1) was the highest (77.0 kDa) among previously reported V-BPOs from Laurencia with a peptide insertion Asn194-Ser221 containing short Gln repeats. It shares approximately 60% amino acid sequence identity with V-BPOs from L. nipponica (LnVBPO1 and LnVBPO2) and L. okamurae (LoVBPO1a and LoVBPO2a). Heterologously expressed LsVBPO1 in Escherichia coli was partially purified and exhibited low but significant bromination activity of 38 U mg(-1) protein using monochlorodimedone. The pH optimum was 8.0, which was more alkaline than that for LnVBPOs and LoVBPO2a (pH 7.0). The K-m for H2O2 was 0.04 mM, comparable to LnVBPO1 (0.026 mM), LnVBPO2 (0.025 mM), and LoVBPO2a (0.014 mM). LsVPBO1 retained its bromination activity until 45 degrees C for 20 min. When incubated at 55 degrees C for 20 min, catalytic activity decreased rapidly, as shown for LnVBPO1 and LoVBPO2a (retained at 45 degrees C, decreased at 55 degrees C) and LnVBPO2 (retained at 55 degrees C, decreased at 65 degrees C). Unlike other V-BPOs from Laurencia (LnVBPO1, LnVBPO2, and LoVBPO2a), dialysis and concentration during purification process were rapidly inactivated LsVBPO1, suggesting its structural instability.
• Spontaneous Cell Lysis by <i>Pelomonas saccharophila</i> MRB3 Provides Plant-Available Macronutrients in Hydroponic Growth Media and Accelerates Biomass Production of Duckweed

  Hidehiro Ishizawa, Yukiko Kaji, Yuki Shimizu, Masashi Kuroda, Daisuke Inoue, Ayaka Makino, Ryosuke Nakai, Hideyuki Tamaki, Masaaki Morikawa, Michihiko Ike

  Journal of Water and Environment Technology 21 (1) 49 - 58 2023/01 [Refereed]
  
• Characterization of vanadium-dependent bromoperoxidases involved in the production of brominated sesquiterpenes by the red alga Laurencia okamurae

  Takafumi Ishikawa, Kenji Washio, Kensuke Kaneko, Xiao Rong Tang, Masaaki Morikawa, Tatsufumi Okino

  Applied Phycology 3 (1) 120 - 131 2022/12/31 [Refereed]
  
• Diazotrophic bacterium Azotobacter vinelandii as a mutualistic growth promoter of an aquatic plant: Lemna minor

  Sajjad Kamal Shuvro, Rahul Jog, Masaaki Morikawa

  Plant Growth Regulation 0167-6903 2022/09/21 [Refereed]
  
• Nostosin G and Spiroidesin B from the Cyanobacterium Dolichospermum sp. NIES-1697

  Chin-Soon Phan, Jakia Jerin Mehjabin, Andrea Roxanne J. Anas, Masahiro Hayasaka, Reiko Onoki, Juting Wang, Taiki Umezawa, Kenji Washio, Masaaki Morikawa, Tatsufumi Okino

  Journal of Natural Products 0163-3864 2022/08/10 [Refereed]
  
• Isolation of Aquatic Plant Growth-Promoting Bacteria for the Floating Plant Duckweed (Lemna minor)

  Ayaka Makino, Ryosuke Nakai, Yasuko Yoneda, Tadashi Toyama, Yasuhiro Tanaka, Xian-Ying Meng, Kazuhiro Mori, Michihiko Ike, Masaaki Morikawa, Yoichi Kamagata, Hideyuki Tamaki

  Microorganisms 10 (8) 1564 - 1564 2022/08/03 [Refereed]
   

  Plant growth-promoting bacteria (PGPB) can exert beneficial growth effects on their host plants. Little is known about the phylogeny and growth-promoting mechanisms of PGPB associated with aquatic plants, although those of terrestrial PGPB have been well-studied. Here, we report four novel aquatic PGPB strains, MRB1–4 (NITE P-01645–P-01648), for duckweed Lemna minor from our rhizobacterial collection isolated from Lythrum anceps. The number of L. minor fronds during 14 days co-culture with the strains MRB1–4 increased by 2.1–3.8-fold, compared with an uninoculated control; the plant biomass and chlorophyll content in co-cultures also increased. Moreover, all strains possessed an indole-3-acetic acid production trait in common with a plant growth-promoting trait of terrestrial PGPB. Phylogenetic analysis showed that three strains, MRB-1, -3, and -4, were affiliated with known proteobacterial genera (Bradyrhizobium and Pelomonas); this report is the first to describe a plant-growth promoting activity of Pelomonas members. The gammaproteobacterial strain MRB2 was suggested to be phylogenetically novel at the genus level. Under microscopic observation, the Pelomonas strain MRB3 was epiphytic and adhered to both the root surfaces and fronds of duckweed. The duckweed PGPB obtained here could serve as a new model for understanding unforeseen mechanisms behind aquatic plant-microbe interactions.
• Complete Genome Sequence of Luteitalea sp. Strain TBR-22.

  Kyosuke Yamamoto, Yasuko Yoneda, Ayaka Makino, Yasuhiro Tanaka, Xian-Ying Meng, Junko Hashimoto, Kazuo Shin-Ya, Noriyuki Satoh, Manabu Fujie, Tadashi Toyama, Kazuhiro Mori, Michihiko Ike, Masaaki Morikawa, Yoichi Kamagata, Hideyuki Tamaki

  Microbiology resource announcements 11 (2) e0045521  2022/02/17 

  We report a complete genome sequence of a novel bacterial isolate, strain TBR-22, belonging to the class Vicinamibacteria of the phylum Acidobacteria, which was isolated from duckweed fronds. The genome expands our knowledge of the lifestyle of this abundant but rarely characterized phylum.
• Draft Genome Sequence of Bryobacteraceae Strain F-183.

  Kyosuke Yamamoto, Yasuko Yoneda, Ayaka Makino, Yasuhiro Tanaka, Xian-Ying Meng, Junko Hashimoto, Kazuo Shin-Ya, Noriyuki Satoh, Manabu Fujie, Tadashi Toyama, Kazuhiro Mori, Michihiko Ike, Masaaki Morikawa, Yoichi Kamagata, Hideyuki Tamaki

  Microbiology resource announcements 11 (1) e0045321  2022/01/20 

  Here, we report a draft genome sequence of a bacterial strain, F-183, isolated from a duckweed frond. Strain F-183 belongs to the family Bryobacteraceae of the phylum Acidobacteria, and its genomic information would contribute to understanding the ecophysiology of this abundant but rarely characterized phylum.
• Growth Promotion of Giant Duckweed Spirodela polyrhiza (Lemnaceae) by Ensifer sp. SP4 Through Enhancement of Nitrogen Metabolism and Photosynthesis

  Tadashi Toyama, Kazuhiro Mori, Yasuhiro Tanaka, Michihiko Ike, Masaaki Morikawa

  MOLECULAR PLANT-MICROBE INTERACTIONS 35 (1) 28 - 38 0894-0282 2022/01 [Refereed]
   

  Duckweeds (Lemnaceae) are representative producers in fresh aquatic ecosystems and also yield sustainable biomass for animal feeds, human foods, and biofuels, and contribute toward effective wastewater treatment; thus, enhancing duckweed productivity is a critical challenge. Plant-growth -promoting bacteria (PGPB) can improve the productivity of terrestrial plants; however, duckweed-PGPB interactions remain unclear and no previous study has investigated the molecular mechanisms underlying duckweed-PGPB interaction. Herein, a PGPB, Ensifer sp. strain SP4, was newly isolated from giant duckweed (Spirodela polyrhiza), and the interactions between S. polyrhiza and SP4 were investigated through physiological, biochemical, and metabolomic analyses. In S. polyrhiza and SP4 coculture, SP4 increased the nitrogen (N), chlorophyll, and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) contents and the photosynthesis rate of S. polyrhiza by 2.5-, 2.5-, 2.7-, and 2.4-fold, respectively. Elevated photosynthesis increased the relative growth rate and biomass productivity of S. polyrhiza by 1.5 and 2.7-fold, respectively. Strain SP4 significantly altered the metabolomic profile of S. polyrhiza, especially its amino acid profile. N stable isotope analysis revealed that organic N compounds were transferred from SP4 to S. polyrhiza. These N compounds, particularly glutamic acid, possibly triggered the increase in photosynthetic and growth activities. Accordingly, we propose a new model for the molecular mechanism underlying S. polyrhiza growth promotion by its associated bacteria Ensifer sp. SP4, which occurs through enhanced N compound metabolism and photosynthesis. Our findings show that Ensifer sp. SP4 is a promising PGPB for increasing biomass yield, wastewater purification activity, and CO2 capture of S. polyrhiza.
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• Construction of a black-dirt formation model using microorganisms obtained from the toilet bowl

  Michitaka Muramatsu, Chie Ito, Kazuma Niizeki, Mitsuhiro Gomi, Masaaki Morikawa

  Journal of Environmental Biotechnology 21 (1) 63 - 66 2021/07 [Refereed][Not invited]
  
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• Novel Plant-Associated Acidobacteria Promotes Growth of Common Floating Aquatic Plants, Duckweeds

  Yasuko Yoneda, Kyosuke Yamamoto, Ayaka Makino, Yasuhiro Tanaka, Xian-Ying Meng, Junko Hashimoto, Kazuo Shin-ya, Noriyuki Satoh, Manabu Fujie, Tadashi Toyama, Kazuhiro Mori, Michihiko Ike, Masaaki Morikawa, Yoichi Kamagata, Hideyuki Tamaki

  MICROORGANISMS 9 (6) 2021/06 [Refereed][Not invited]
   

  Duckweeds are small, fast growing, and starch- and protein-rich aquatic plants expected to be a next generation energy crop and an excellent biomaterial for phytoremediation. Despite such an importance, very little is known about duckweed-microbe interactions that would be a key biological factor for efficient industrial utilization of duckweeds. Here we first report the duckweed growth promoting ability of bacterial strains belonging to the phylum Acidobacteria, the members of which are known to inhabit soils and terrestrial plants, but their ecological roles and plant-microbe interactions remain largely unclear. Two novel Acidobacteria strains, F-183 and TBR-22, were successfully isolated from wild duckweeds and phylogenetically affiliated with subdivision 3 and 6 of the phylum, respectively, based on 16S rRNA gene sequence analysis. In the co-culture experiments with aseptic host plants, the F-183 and TBR-22 strains visibly enhanced growth (frond number) of six duckweed species (subfamily Lemnoideae) up to 1.8-5.1 times and 1.6-3.9 times, respectively, compared with uninoculated controls. Intriguingly, both strains also increased the chlorophyll content of the duckweed (Lemna aequinoctialis) up to 2.4-2.5 times. Under SEM observation, the F-183 and TBR-22 strains were epiphytic and attached to the surface of duckweed. Taken together, our findings suggest that indigenous plant associated Acidobacteria contribute to a healthy growth of their host aquatic plants.
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• Indigenous bacteria, an excellent reservoir of functional plant growth promoters for enhancing duckweed biomass yield on site

  Yeni Khairina, Rahul Jog, Chanita Boonmak, Tadashi Toyama, Tokitaka Oyama, Masaaki Morikawa

  Chemosphere 268 129247 - 129247 0045-6535 2021/04 [Refereed][Not invited]
  
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• A cyclic lipopeptide surfactin is a species-selective Hsp90 inhibitor that suppresses cyanobacterial growth

  Hitoshi Nakamoto, Yuhei Yokoyama, Takahiro Suzuki, Yuri Miyamoto, Takashi Fujishiro, Masaaki Morikawa, Yoshihiko Miyata

  The Journal of Biochemistry 170 (2) 255 - 264 0021-924X 2021/03/26 [Refereed][Not invited]
   

  Heat shock protein 90 (Hsp90) is essential for eukaryotic cells, whereas bacterial homologs play a role under stresses and in pathogenesis. Identifying species-specific Hsp90 inhibitors is challenging because Hsp90 is evolutionarily conserved. We found that a cyclic lipopeptide surfactin inhibits the ATPase activity of Hsp90 from the cyanobacterium Synechococcus elongatus (S.elongatus) PCC 7942 but does not inhibit Escherichia coli (E.coli), yeast and human Hsp90s. Molecular docking simulations indicated that surfactin could bind to the N-terminal dimerization interface of the cyanobacterial Hsp90 in the ATP- and ADP-bound states, which provided molecular insights into the species-selective inhibition. The data suggest that surfactin inhibits a rate-limiting conformational change of S.elongatus Hsp90 in the ATP hydrolysis. Surfactin also inhibited the interaction of the cyanobacterial Hsp90 with a model substrate, and suppressed S.elongatus growth under heat stress, but not that of E.coli. Surfactin did not show significant cellular toxicity towards mammalian cells. These results indicate that surfactin inhibits the cellular function of Hsp90 specifically in the cyanobacterium. The present study shows that a cyclic peptide has a great specificity to interact with a specific homolog of a highly conserved protein family.
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• Multiple biosurfactant production by Aureobasidium pullulans strain YTP6-14 in aqueous and heavy oil layers.

  Natwara Amatyakul, Suthep Thaniyavarn, Masaaki Morikawa, Jiraporn Thaniyavarn

  The Journal of general and applied microbiology 66 (6) 330 - 338 2021/02/26 [Refereed][Not invited]
   

  Aureobasidium pullulans YTP6-14 was demonstrated to be an excellent multiple biosurfactant producer utilizing cheap carbon sources available in Thailand, including glycerol and cassava flour hydrolysate. A. pullulans YTP6-14 maximally produced 1.81 g/l biosurfactant in an aqueous layer (BS-AQ) in a medium containing glycerol, and 7.37 or 6.37 g/l biosurfactant in a heavy oil layer (BS-HO) in cassava flour hydrolysate or a glucose containing medium, respectively. Each BS-AQ and BS-HO had critical micelle concentration values of 41.32 mg/l and 13.51 mg/l, and both biosurfactants formed a stable food oil emulsion and reduced the amount of biofilms formed by Streptococcus sobrinus and Streptococcus mutans. BS-AQ and BS-HO were mainly composed of liamocins or exophilins and massoia lactone, respectively.
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• Isolation and Characterization of Novel Plant Growth-Promoting Bacteria from the Fronds of Duckweed

  TOMOKI IWASHITA, YASUHIRO TANAKA, HIDEYUKI TAMAKI, RYOSUKE NAKAI, YASUKO YONEDA, AYAKA MAKINO, TADASHI TOYAMA, YOICHI KAMAGATA, MASAAKI MORIKAWA, KAZUHIRO MORI

  Japanese Journal of Water Treatment Biology 57 (1) 1 - 9 0910-6758 2021 [Refereed][Not invited]
  
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• Community dynamics of duckweed-associated bacteria upon inoculation of plant growth-promoting bacteria

  Hidehiro Ishizawa, Masashi Kuroda, Daisuke Inoue, Masaaki Morikawa, Michihiko Ike

  FEMS Microbiology Ecology 96 (7) 0168-6496 2020/07/01 [Refereed][Not invited]
   

  Plant growth-promoting bacteria (PGPB) have recently been demonstrated as a promising agent to improve wastewater treatment and biomass production efficiency of duckweed hydrocultures. With a view to their reliable use in aqueous environments, this study analysed the plant colonization dynamics of PGPB and the ecological consequences for the entire duckweed-associated bacterial community. A PGPB strain, Aquitalea magnusonii H3, was inoculated to duckweed at different cell densities or timings in the presence of three environmental bacterial communities. The results showed that strain H3 improved duckweed growth by 11.7–32.1% in five out of nine experiments. Quantitative-PCR and amplicon sequencing analyses showed that strain H3 successfully colonized duckweed after 1 and 3 d of inoculation in all cultivation tests. However, it significantly decreased in number after 7 d, and similar bacterial communities were observed on duckweed regardless of H3 inoculation. Predicted metagenome analysis suggested that genes related to bacterial chemotactic motility and surface attachment systems are consistently enriched through community assembly on duckweed. Taken together, strain H3 dominantly colonized duckweed for a short period and improved duckweed growth. However, the inoculation of the PGPB did not have a lasting impact due to the strong resilience of the natural duckweed microbiome.
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• Biosurfactants from Marine Cyanobacteria Collected in Sabah, Malaysia

  Jakia Jerin Mehjabin, Liang Wei, Julie G. Petitbois, Taiki Umezawa, Fuyuhiko Matsuda, Charles S. Vairappan, Masaaki Morikawa, Tatsufumi Okino

  Journal of Natural Products 83 (6) 1925 - 1930 0163-3864 2020/06/26 [Refereed][Not invited]
   

  Chemical investigation of the organic extract from Moorea bouillonii, collected in Sabah, Malaysia, led to the isolation of three new chlorinated fatty acid amides, columbamides F (1), G (2), and H (3). The planar structures of 1-3 were established by a combination of mass spectrometric and NMR spectroscopic analyses. The absolute configuration of 1 was determined by Marfey's analysis of its hydrolysate and chiral-phase HPLC analysis after conversion and esterification with Ohrui's acid, (1S,2S)-2-(anthracene-2,3-dicarboximido)cyclohexanecarboxylic acid. Compound 1 showed biosurfactant activity by an oil displacement assay. Related known fatty acid amides columbamide D and serinolamide C exhibited biosurfactant activity with critical micelle concentrations of about 0.34 and 0.78 mM, respectively.
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• Enhanced lipid productivity of Chlamydomonas reinhardtii with combination of NaCl and CaCl2 stresses

  Le Thai Hang, Kazuhiro Mori, Yasuhiro Tanaka, Masaaki Morikawa, Tadashi Toyama

  Bioprocess and Biosystems Engineering 43 (6) 971 - 980 1615-7591 2020/06 [Refereed][Not invited]
  
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• Antimicrobial electrospun fiber mat from gelatin and crude extract lipopeptides

  Tipnipa Yoochang, Jirarat Anuntagool, Suthep Thaniyavarn, Masaaki Morikawa, Jiraporn Thaniyavarn

  Songklanakarin Journal of Science and Technology 42 (1) 101 - 108 2020/01 [Refereed][Not invited]
  
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• Enhanced biomass production and nutrient removal capacity of duckweed via two-step cultivation process with a plant growth-promoting bacterium, Acinetobacter calcoaceticus P23

  Hidehiro Ishizawa, Yuka Ogata, Yoshiyuki Hachiya, Ko-ichiro Tokura, Masashi Kuroda, Daisuke Inoue, Tadashi Toyama, Yasuhiro Tanaka, Kazuhiro Mori, Masaaki Morikawa, Michihiko Ike

  Chemosphere 238 124682 - 124682 0045-6535 2020/01 [Refereed][Not invited]
   

  © 2019 Elsevier Ltd Plant growth-promoting bacteria (PGPB) are considered a promising tool to improve biomass production and water remediation by the aquatic plant, duckweed; however, no effective methodology is available to utilize PGPB in large hydroponic systems. In this study, we proposed a two-step cultivation process, which comprised of a “colonization step” and a “mass cultivation step,” and examined its efficacy in both bucket-scale and flask-scale cultivation experiments. We showed that in the outdoor bucket-scale experiments using three kinds of environmental water, plants cultured through the two-step cultivation method with the PGPB strain, Acinetobacter calcoaceticus P23, yielded 1.9 to 2.3 times more biomass than the control (without PGPB inoculation). The greater nitrogen and phosphorus removals compared to control were also attained, indicating that this strategy is useful for accelerating nutrient removal by duckweed. Flask-scale experiments using non-sterile pond water revealed that inoculation of strain P23 altered duckweed surface microbial community structures, and the beneficial effects of the inoculated strain P23 could last for 5–10 d. The loss of the duckweed growth-promoting effect was noticeable when the colonization of strain P23 decreased in the plant. These observations suggest that the stable colonization of the plant with PGPB is the key for maintaining the accelerated duckweed growth and nutrient removal in this cultivation method. Overall, our results suggest the possibility of an improved duckweed production using a two-step cultivation process with PGPB.
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• Enhanced production of biomass and lipids by Euglena gracilis via co-culturing with a microalga growth-promoting bacterium, Emticicia sp. EG3

  Tadashi Toyama, Tsubasa Hanaoka, Koji Yamada, Kengo Suzuki, Yasuhiro Tanaka, Masaaki Morikawa, Kazuhiro Mori

  Biotechnology for Biofuels 12 (1) 205 - 205 2019/12 [Refereed][Not invited]
   

  Euglena gracilis, a unicellular flagellated microalga, is regarded as one of the most promising species as microalgal feedstock for biofuels. Its lipids (mainly wax esters) are suitable for biodiesel and jet fuel. Culture ofE. gracilisusing wastewater effluent will improve the economics ofE. gracilisbiofuel production. Enhancement of the productivity ofE. gracilisbiomass is critical to creating a highly efficient biofuels production system. Certain bacteria have been found to promote microalgal growth by creating a favorable microenvironment. These bacteria have been characterized as microalgae growth-promoting bacteria (MGPB). Co-culture of microalgae with MGPB might offer an effective strategy to enhance microalgal biomass production in wastewater effluent culture systems. However, no MGPB has been identified to enhance the growth ofE. gracilis. The objectives of this study were, therefore, to isolate and characterize the MGPB effective forE. gracilisand to demonstrate that the isolated MGPB indeed enhances the production of biomass and lipids byE. gracilisin wastewater effluent culture system. A bacterium,Emticiciasp. EG3, which is capable of promoting the growth of microalgaE. gracilis, was isolated from anE. gracilis-municipal wastewater effluent culture. Biomass production rate ofE. graciliswas enhanced 3.5-fold and 3.1-fold by EG3 in the co-culture system using a medium of heat-sterilized and non-sterilized wastewater effluent, respectively, compared to growth in the same effluent culture but without EG3. Two-step culture system was examined as follows:E. graciliswas cultured with or without EG3 in wastewater effluent in the first step and was further grown in wastewater effluent in the second step. Production yields of biomass and lipids byE. graciliswere enhanced 3.2-fold and 2.9-fold, respectively, in the second step of the system in whichE. graciliswas co-cultured with EG3 in the first step. Emticiciasp. EG3 is the first MGPB forE. gracilis. Growth-promoting bacteria such as EG3 will be promising agents for enhancingE. gracilisbiomass/biofuel productivities.
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• Colonization and Competition Dynamics of Plant Growth-Promoting/Inhibiting Bacteria in the Phytosphere of the Duckweed Lemna minor

  Hidehiro Ishizawa, Masashi Kuroda, Kanako Inoue, Daisuke Inoue, Masaaki Morikawa, Michihiko Ike

  Microbial Ecology 77 (2) 440 - 450 0095-3628 2019/02/15 [Refereed][Not invited]
   

  © 2019, Springer Science+Business Media, LLC, part of Springer Nature. Despite the considerable role of aquatic plant-associated bacteria in host plant growth and nutrient cycling in aquatic environments, the mode of their plant colonization has hardly been understood. This study examined the colonization and competition dynamics of a plant growth-promoting bacterium (PGPB) and two plant growth-inhibiting bacteria (PGIB) in the aquatic plant Lemna minor (common duckweed). When inoculated separately to L. minor, each bacterial strain quickly colonized at approximately 10 6 cells per milligram (plant fresh weight) and kept similar populations throughout the 7-day cultivation time. The results of two-membered co-inoculation assays revealed that the PGPB strain Aquitalea magnusonii H3 consistently competitively excluded the PGIB strain Acinetobacter ursingii M3, and strain H3 co-existed at almost 1:1 proportion with another PGIB strain, Asticcacaulis excentricus M6, regardless of the inoculation ratios (99:1–1:99) and inoculation order. We also found that A. magnusonii H3 exerted its growth-promoting effect over the negative effects of the two PGIB strains even when only a small amount was inoculated, probably due to its excellent competitive colonization ability. These experimental results demonstrate that there is a constant ecological equilibrium state involved in the bacterial colonization of aquatic plants.
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• Biomass production and nutrient removal through cultivation of Euglena gracilis in domestic wastewater

  Masashi Kuroda, Taro Horino, Daisuke Inoue, Masaaki Morikawa, Michihiko Ike

  Japanese Journal of Water Treatment Biology 54 (4) 105 - 113 2018/12 [Refereed][Not invited]
  
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• Effects of co-inoculation of two different plant growth-promoting bacteria on duckweed

  Yusuke Yamakawa, Rahul Jog, Masaaki Morikawa

  Plant Growth Regulation 86 (2) 287 - 296 2018/11/06 [Refereed][Not invited]
  
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• Effect of Exogenous General Plant Growth Regulators on the Growth of the Duckweed Lemna minor

  Utami, Desi, Kawahata, Ami, Sugawara, Masayuki, Jog, Rahul N., Miwa, Kyoko, Morikawa, Masaaki

  Frontiers in Chemistry 6 251  2018/07 [Refereed][Not invited]
  
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• Growth promotion of three microalgae, Chlamydomonas reinhardtii, Chlorella vulgaris and Euglena gracilis, by in situ indigenous bacteria in wastewater effluent

  Toyama, Tadashi, Kasuya, Mari, Hanaoka, Tsubasa, Kobayashi, Naoto, Tanaka, Yasuhiro, Inoue, Daisuke, Sei, Kazunari, Morikawa, Masaaki, Mori, Kazuhiro

  Biotechnology For Biofuels 11 176  2018/06 [Refereed][Not invited]
  
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• Comprehensive evaluation of nitrogen removal rate and biomass, ethanol, and methane production yields by combination of four major duckweeds and three types of wastewater effluent

  Tadashi Toyama, Tsubasa Hanaoka, Yasuhiro Tanaka, Masaaki Morikawa, Kazuhiro Mori

  Bioresource Technology 250 464 - 473 1873-2976 2018/02/01 [Refereed][Not invited]
   

  To assess the potential of duckweeds as agents for nitrogen removal and biofuel feedstocks, Spirodela polyrhiza, Lemna minor, Lemna gibba, and Landoltia punctata were cultured in effluents of municipal wastewater, swine wastewater, or anaerobic digestion for 4 days. Total dissolved inorganic nitrogen (T-DIN) of 20–50 mg/L in effluents was effectively removed by inoculating with 0.3–1.0 g/L duckweeds. S. polyrhiza showed the highest nitrogen removal (2.0–10.8 mg T-DIN/L/day) and biomass production (52.6–70.3 mg d.w./L/day) rates in all the three effluents. Ethanol and methane were produced from duckweed biomass grown in each effluent. S. polyrhiza and L. punctata biomass showed higher ethanol (0.168–0.191, 0.166–0.172 and 0.174–0.191 g-ethanol/g-biomass, respectively) and methane (340–413 and 343–408 NL CH4/kg VS, respectively) production potentials than the others, which is related to their higher carbon and starch contents and calorific values.
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• Production of massoia lactone by Aureobasidium pullulans YTP6-14 isolated from the Gulf of Thailand and its fragrant biosurfactant properties

  S. Luepongpattana, J. Thaniyavarn, M. Morikawa

  JOURNAL OF APPLIED MICROBIOLOGY 123 (6) 1488 - 1497 1364-5072 2017/12 [Refereed][Not invited]
   

  Aims: In order to add to the existing knowledge about structural diversity of biosurfactants, marine environment was chosen to discover a new type of biosurfactant-producing fungus. Methods and Results: A number of fungi were collected from the Gulf of Thailand and examined for biosurfactant productivities. A dimorphic fungus, Aureobasidium pullulans YTP6-14, produced several different biosurfactants in both heavy oil and aqueous layers of the culture. Surface tension of the aqueous layer was decreased to 31.4 mN m(-1) and oil displacement area reached 53 cm(2)/10 mu l after 7 days of cultivation. Critical micelle concentration and minimum surface tension values of the crude biosurfactants prepared from the aqueous layer were 39 mg l(-1) and 31.6 mN m(-1) respectively. Surface tension values remained unchanged over a wide range of pII and NaCl concentrations, suggesting their nonionic feature. LC/MS and NMR analyses revealed that one of the main active compounds in the aqueous layer was 5-hydroxy-2-decenoic acid delta-lactone, known as massoia lactone. Massoia lactone indeed showed significant surface tension reduction capacity of 43.3 mN m(-1) at 1 mg ml(-1). Significance and Impact of the Study: This is the first report for the production of a fragrant biosurfactant, massoia lactone by a fungus A. pullulans. Massoia lactone has been industrially prepared from aromatic bark of an endangered tree species, Cryptocarya massoy, growing in rainforests. This report expands the diversity of biosurfactants produced by A. pullulans and also points to its possibility in contributing to the green sustainable chemistry, and ultimately rainforest conservation.
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• Differential oxidative and antioxidative response of duckweed Lemna minor toward plant growth promoting/inhibiting bacteria

  Hidehiro Ishizawa, Masashi Kuroda, Masaaki Morikawa, Michihiko Ike

  PLANT PHYSIOLOGY AND BIOCHEMISTRY 118 667 - 673 0981-9428 2017/09 [Refereed][Not invited]
   

  Bacteria colonizing the plant rhizosphere are believed to positively or negatively affect the host plant productivity. This feature has inspired researchers to engineer such interactions to enhance crop production. However, it remains to be elucidated whether rhizobacteria influences plant oxidative stress visa-vis other environmental stressors, and whether such influence is associated with their growth promoting/inhibiting ability. In this study, two plant growth-promoting bacteria (PGPB) and two plant growth-inhibiting bacteria (PGIB) were separately inoculated into axenic duckweed (Lemna minor) culture under laboratory conditions for 4 and 8 days in order to investigate their effects on plant oxidative stress and antioxidant activities. As previously characterized, the inoculation of PGPB and PGIB strains accelerated and reduced the growth of L. minor, respectively. After 4 and 8 days of cultivation, compared to the PGPB strains, the PGIB strains induced larger amounts of O-2(center dot-), H2O2, and malondialdehyde (MDA) in duckweed, although all bacterial strains consistently increased O-2(center dot-) content by two times more than that in the aseptic control plants. Activities of five antioxidant enzymes were also elevated by the inoculation of PGIB, confirming the severe oxidative stress condition in plants. These results suggest that the surface attached bacteria affect differently on host oxidative stress and its response, which degree correlates negatively to their effects on plant growth. (C) 2017 Elsevier Masson SAS. All rights reserved.
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• Enhanced biomass production of duckweeds by inoculating a plant growth-promoting bacterium, Acinetobacter calcoaceticus P23, in sterile medium and non-sterile environmental waters

  T. Toyama, M. Kuroda, Y. Ogata, Y. Hachiya, A. Quach, K. Tokura, Y. Tanaka, K. Mori, M. Morikawa, M. Ike

  WATER SCIENCE AND TECHNOLOGY 76 (6) 1418 - 1428 0273-1223 2017/09 [Refereed][Not invited]
   

  Duckweed offers the promise of a co-benefit culture combining water purification with biomass production. Acinetobacter calcoaceticus P23 is a plant growth-promoting bacterium isolated from a duckweed, Lemna aequinoctialis. This study quantified its growth-promoting effect on three duckweeds (L. aoukikusa, L. minor, and Spirodela polyrhiza) in sterile Hoagland solution and evaluated its usefulness in duckweed culture under non-sterile conditions. P23 promoted growth of three duckweeds in sterile Hoagland solution at low to high nutrient concentrations (1.25-10 mg NO3-N/L and 0.25-2.0 mg PO4-P/L). It increased the biomass production of L. aequinoctialis 3.8-4.3-fold, of L. minor 2.3-3.3-fold, and of S. polyrhiza 1.4-1.5-fold after 7 days compared with noninoculated controls. P23 also increased the biomass production of L. minor 2.4-fold in pond water and 1.7-fold in secondary effluent of a sewage treatment plant under non-sterile conditions at laboratory-scale experiments. P23 rescued L. minor from growth inhibition caused by microorganisms indigenous to the pond water. The results demonstrate that the use of P23 in duckweed culture can improve the efficiency of duckweed biomass production, and a positive effect of P23 on duckweed-based wastewater treatment can be assumed.
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• Evaluation of environmental bacterial communities as a factor affecting the growth of duckweed Lemna minor

  Hidehiro Ishizawa, Masashi Kuroda, Masaaki Morikawa, Michihiko Ike

  BIOTECHNOLOGY FOR BIOFUELS 10 62  1754-6834 2017/03 [Refereed][Not invited]
   

  Background: Duckweed (family Lemnaceae) has recently been recognized as an ideal biomass feedstock for biofuel production due to its rapid growth and high starch content, which inspired interest in improving their productivity. Since microbes that co-exist with plants are known to have significant effects on their growth according to the previous studies for terrestrial plants, this study has attempted to understand the plant-microbial interactions of a duckweed, Lemna minor, focusing on the growth promotion/inhibition effects so as to assess the possibility of accelerated duckweed production by modifying co-existing bacterial community. Results: Co-cultivation of aseptic L. minor and bacterial communities collected from various aquatic environments resulted in changes in duckweed growth ranging from -24 to + 14% compared to aseptic control. A number of bacterial strains were isolated from both growth-promoting and growth-inhibitory communities, and examined for their co-existing effects on duckweed growth. Irrespective of the source, each strain showed promotive, inhibitory, or neutral effects when individually co-cultured with L. minor. To further analyze the interactions among these bacterial strains in a community, binary combinations of promotive and inhibitory strains were co-cultured with aseptic L. minor, resulting in that combinations of promotive-promotive or inhibitory-inhibitory strains generally showed effects similar to those of individual strains. However, combinations of promotive-inhibitory strains tended to show inhibitory effects while only Aquitalea magnusonii H3 exerted its plant growth-promoting effect in all combinations tested. Conclusion: Significant change in biomass production was observed when duckweed was co-cultivated with environmental bacterial communities. Promotive, neutral, and inhibitory bacteria in the community would synergistically determine the effects. The results indicate the possibility of improving duckweed biomass production via regulation of co-existing bacterial communities.
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• Production of biosurfactant by Wickerhamomyces anomalus PY189 and its application in lemongrass oil encapsulation

  Chanapa Dejwatthanakomol, Jirarat Anuntagool, Masaaki Morikawa, Jiraporn Thaniyavarn

  SCIENCEASIA 42 (4) 252 - 258 1513-1874 2016/08 [Refereed][Not invited]
   

  In this study, the production and characterization of a biosurfactant from yeast Wickerhamomyces anomalus strain PY189 was carried out. The highest efficiency for biosurfactant production was found when the organism was grown in a medium containing 4% (v/v) soya bean oil and 0.4% (w/v) NaNO3 at 30 degrees C and pH 5.5 for 7 days. After 7 days of cultivation, W. anomalus PY189 was able to produce up to 0.57 g/l of biosurfactant as ethyl acetate extracts. The culture supernatant was able to reduce the surface tension of the culture broth from 42.5 mN/mto 36.5 mN/m with a critical micelle concentration of 204 mg/l. The crude extract of biosurfactants was then applied to the encapsulation of lemongrass oil. Emulsions of lemongrass oil in 20 mg/dl maltodextrin solution (oil: maltodextrin solution ratios of 0.2:1, 0.15:1, and 0.1:1) containing 0.8% and 1% (w/v) crude biosurfactant extract were stable for at least 24 h and had an average oil droplet size of less than 10 mu m. Lemongrass oil microcapsules were later produced using a spray drying technique. This microcapsule exhibited microbial growth inhibition activity against E. coli, S. aureus, and Salmonella at 5% (w/v) concentration.
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• Wewakazole B, a Cytotoxic Cyanobactin from the Cyanobacterium Moorea producens Collected in the Red Sea

  Julius Adam V. Lopez, Sultan S. Al-Lihaibi, Walied M. Alarif, Ahmed Abdel-Lateff, Yasuyuki Nogata, Kenji Washio, Masaaki Morikawa, Tatsufumi Okino

  JOURNAL OF NATURAL PRODUCTS 79 (4) 1213 - 1218 0163-3864 2016/04 [Refereed][Not invited]
   

  A mass spectrometry (MS)-guided isolation has led to the purification of a new cyanobactin, wewakazole B (1), along with the known compound curacin D from a Red Sea Moorea producens. The planar structure of 1 was elucidated using a combination of NMR and MS techniques. After ozonolysis and acid hydrolysis, the absolute configurations of the amino acid components of 1 were determined by chiral-phase LC-MS and HPLC analyses. Notably, compound 1 exhibited cytotoxic activity toward human MCF7 breast cancer cells (IC50 = 0.58 mu M) and human H460 lung cancer cells (IC50 = 1.0 mu M) and was also found to be inactive in a siderophore assay.
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• Isolation and characterization of an early colonizing Rhizobium sp. R8 from a household toilet bowl

  Toru Fukano, Mitsuhiro Gomi, Yukihiko Osaki, Masaaki Morikawa

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 79 (7) 1207 - 1215 0916-8451 2015/07 [Refereed][Not invited]
   

  The bacterial community structure was compared between the third days', one week', and three weeks' biofilm samples from the surface of a household toilet bowl. It was found that the PCR-DGGE band pattern of 16S rRNA gene was dramatically changed after the third day and was not further changed until three weeks. This result suggests that there are early and late colonizing bacterial groups. One of the early colonizers isolated from the third days' sample was Rhizobium sp. R8, a closest relative to Rhizobium giardinii, which exhibited the highest biofilm formation activity in an artificial urine condition. R8 produced extracellular polysaccharides containing galactose, glucose, and mannose at the molar ratio of 8:1:1, which were probably responsible for the biofilm formation. Its excelled biofilm formation and urease activities together with the lack of nodulation and nitrogen fixing genes in R8 suggest that this strain has been specifically adapted to urine condition in a toilet bowl.
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• Wewakazole B, A New Cyclic Dodecapeptide from the Red Sea Cyanobacterium Moorea producens

  Lopez Julius Adam Velasco, Al-Lihaibi Sultan S., Alarif Walied M., Abdel-Lateff Ahmed, Washio Kenji, Morikawa Masaaki, Okino Tatsufumi

  Symposium on the Chemistry of Natural Products, symposium papers 天然有機化合物討論会実行委員会 57 PosterP23  2015 

  Cyanobacteria are prolific sources of novel bioactive compounds. The chemical profiles we obtained using LC-MS and checked with the MarinLit database showed that there are potential new compounds from Moorea producens samples obtained from the Red Sea. In this presentation the isolation and structure elucidation of a new cyclic dodecapeptide, wewakazole B (1), is reported. The brownish-red filamentous sample was obtained by SCUBA near Jeddah, Saudi Arabia. A small portion of the sample was preserved in RNAlater and identified as M. producens by standard 16S rRNA gene sequencing using cyanobacteria-specific primers 106F and 1509R, and internal primers 359F and 781R (Martinez-Murcia et al., 1995; Nubel et al., 1997). The bulk of the sample was extracted with methanol followed by solvent partition using EtOAc and water, then, BuOH. These extracts were tested for antifouling activity against the barnacle larvae Amphibalanus amphitrite, cytotoxicity against MCF-7 breast cancer cells, and trypsin inhibition. LC-MS was carried out in parallel for dereplication in conjunction with the MarinLit database. The extracts were found to be inactive but a unique [M+H]⁺ m/z of 1127 was detected in the EtOAc extract and was pursued. A series of open column chromatography and HPLCs afforded 0.4 mg of 1. The molecular formula, C₅₈H₇₀N₁₂O₁₂, determined by HRESIMS ([M+H]⁺ m/z1127.5310, ∆ = 0.39 ppm), suggested thirty unsaturations. Dereplication using mass and ¹H NMR data showed wewakazole (Nogle et al., 2003) as the nearest candidate. HSQC, COSY, and TOCSY revealed nine common and three modified amino acid residues which were connected by HMBC and ROESY correlations acquiring five fragments. Connections to the oxazole and methyloxazole fragments proved to be a challenge due to the lack of correlations towards neighboring amino acids. All possible combinations were considered that lead to the most logical planar structure and cyclic sequence. Chiral LC-MS and HPLC analysis showed that all amino acid residues possess the L- configuration. The wewakazoles are the only cyclic peptides from marine cyanobacteria having both Oxz and MeOxz. Their function and bioactivity require further investigation.
• Draft genome sequence of acinetobacter calcoaceticus strain P23, a plant growth-promoting bacterium of duckweed

  Masayuki Sugawara, Akira Hosoyama, Atsushi Yamazoe, Masaaki Morikawa

  Genome Announcements 3 (1) e00026-15  2169-8287 2015 [Refereed][Not invited]
   

  Acinetobacter calcoaceticus strain P23 is a plant growth-promoting bacterium, which was isolated from the surface of duckweed. We report here the draft genome sequence of strain P23. The genome data will serve as a valuable reference for understanding the molecular mechanism of plant growth promotion in aquatic plants.
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• cDNA cloning and characterization of vanadium-dependent bromoperoxidases from the red alga Laurencia nipponica

  Kensuke Kaneko, Kenji Washio, Taiki Umezawa, Fuyuhiko Matsuda, Masaaki Morikawa, Tatsufumi Okino

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 78 (8) 1310 - 1319 0916-8451 2014/08 [Refereed][Not invited]
   

  The marine red alga genus Laurencia is one of the richest producers of unique brominated compounds in the marine environment. The cDNAs for two Laurencia nipponica vanadium-dependent bromoperoxidases (LnVBPO1 and LnVBPO2) were cloned and expressed in Escherichia coli. Enzyme assays of recombinant LnVBPO1 and LnVBPO2 using monochlorodimedone revealed that they were thermolabile but their K-m values for Br- were significantly lower than other red algal VBPOs. The bromination reaction was also assessed using laurediol, the predicted natural precursor of the brominated ether laurencin. Laurediol, protected by trimethylsilyl at the enyne, was converted to deacetyllaurencin by the LnVBPOs, which was confirmed by tandem mass spectrometry. Native LnVBPO partially purified from algal bodies was active, suggesting that LnVBPO is functional in vivo. These results contributed to our knowledge of the biosynthesis of Laurencia brominated metabolites.
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• Plant growth-promoting bacterium Acinetobacter calcoaceticus P23 increases the chlorophyll content of the monocot Lemna minor (duckweed) and the dicot Lactuca sativa (lettuce)

  Wakako Suzuki, Masayuki Sugawara, Kyoko Miwa, Masaaki Morikawa

  JOURNAL OF BIOSCIENCE AND BIOENGINEERING 118 (1) 41 - 44 1389-1723 2014/07 [Refereed][Not invited]
   

  Acinetobacter cakoaceticus P23 is a plant growth-promoting bacterium that was isolated from the surface of duckweed (Lemna aoubilmsa). The bacterium was observed to colonize on the plant surfaces and increase the chlorophyll content of not only the monocotyledon Lemna minor but also the dicotyledon Lactuca sativa in a hydroponic culture. This effect on the Lactuca sativa was significant in nutrient-poor ( x 1/100 dilution of H2 medium) and not nutrient-rich ( x 1 or x1/l0 dilutions of H2 medium) conditions. Strain P23 has the potential to play a part in the future development of fertilizers and energy-saving hydroponic agricultural technologies. (C) 2013, The Society for Biotechnology, Japan. All rights reserved.
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• Transformation of iso-pentylbenzene by a biofilm-forming strain of Candida viswanathii TH1 isolated from oil-polluted sediments collected in coastal zones in Vietnam

  Le Thi Nhi Cong, Cung Thi Ngoc Mai, Masaaki Morikawa, Nghiem Ngoc Minh

  JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH PART A-TOXIC/HAZARDOUS SUBSTANCES & ENVIRONMENTAL ENGINEERING 49 (7) 777 - 786 1093-4529 2014/06 [Refereed][Not invited]
   

  This work is aimed to assess the aerobic biotransformation of a branched side chain alkylbenzene, iso-pentylbenzene, by Candida viswanathii TH1. The yeast Candida viswanathii TH1 isolated from oil-polluted sediments collected in coastal zones in Vietnam exhibited as a strain that could better transform branched aromatic hydrocarbons in biofilm (pellicle) than in planktonic form. During incubation of TH1 as biofilm with iso-pentylbenzene, the seven intermediates produced were benzoic acid, phenylacetic acid, 2-methyl-4-phenyl-butan-1-ol, 2-hydroxy-phenylacetic acid, 2-methyl-4-phenylbutyric acid, succinic acid and iso-valerophenone as revealed by gas chromatography/mass spectra and high-performance liquid chromatography analyses. The occurrence of these intermediates showed that iso-pentylbenzene could be oxidized not only via mono- but also by a sub-terminal oxidation pathway. This is the first study on iso-pentylbenzene transformation by a biofilm-forming Candida viswanathii strain. The catabolic versatility of the biofilm-forming strain TH1 and its use for mono and sub-terminal oxidation during the transformation of iso-pentylbenzene enhance our understanding of the degradation of branched side chain phenylalkanes and give new insight into the potential role of such species in the transformation of other recalcitrant aromatic compounds.
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• Cloning and expression of three ladA-type alkane monooxygenase genes from an extremely thermophilic alkane-degrading bacterium Geobacillus thermoleovorans B23

  Chanita Boonmak, Yasunori Takahashi, Masaaki Morikawa

  EXTREMOPHILES 18 (3) 515 - 523 1431-0651 2014/05 [Refereed][Not invited]
   

  An extremely thermophilic bacterium, Geobacillus thermoleovorans B23, is capable of degrading a broad range of alkanes (with carbon chain lengths ranging between C11 and C32) at 70 A degrees C. Whole-genome sequence analysis revealed that unlike most alkane-degrading bacteria, strain B23 does not possess an alkB-type alkane monooxygenase gene. Instead, it possesses a cluster of three ladA-type genes, ladA alpha(B23), ladA beta(B23), and ladB (B23), on its chromosome, whose protein products share significant amino acid sequence identities, 49.8, 34.4, and 22.7 %, respectively, with that of ladA alkane monooxygenase gene found on a plasmid of Geobacillus thermodetrificans NG 80-2. Each of the three genes, ladA alpha(B23), ladA beta(B23), and ladB (B23), was heterologously expressed individually in an alkB1 deletion mutant strain, Pseudomonas fluorescens KOB2 Delta 1. It was found that all three genes were functional in P. fluorescens KOB2 Delta 1, and partially restored alkane degradation activity. In this study, we suggest that G. thermoleovorans B23 utilizes multiple LadA-type alkane monooxygenases for the degradation of a broad range of alkanes.
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• Bromoperoxidases from Laurencia species

  Kaneko Kensuke, Washio Kenji, Umezawa Taiki, Kobayashi Daiki, Tang Xiaorong, Matsuda Fuyuhiko, Morikawa Masaaki, Okino Tatsufumi

  Symposium on the Chemistry of Natural Products, symposium papers 天然有機化合物討論会実行委員会 56 Poster53  2014 

  The genus of red alga Laurencia is one of the richest producers of halogenated compounds (mostly brominated) ³⁾. Murai et al ⁴⁾ and Butler et al ⁹⁾ showed enzymatic bromination of Laurencia C₁₅acetogenins including laurencin (1) ^1,2)and sesquiterpenoids snyderols using partially purified bromoperoxidase (BPO) from L. nipponica and vanadium-dependent BPO (VBPO) from a mixture of red algae including L. pacifica. However, since the lack of information of sequences, the structural information and biochemical aspects of BPO from L. nipponica and VBPO from L. pacifica remains unknown. Here, we report cDNA cloning of VBPOs from L. nipponicaand in vitro enzymatic bromination of putative biosynthetic precursors using recombinant enzymes. Two VBPOs (LnVBPO1 and LnVBPO2) were cloned from L. nipponica and recombinant LnVBPOs were prepared. Recombinant LnVBPOs were subjected to enzyme activity assay. A general substrate, monochlorodimedone (MCD), was utilized for K_m determination for H₂O₂ and , essential for bromination of organic substrate. K_m for H₂O₂was 0.03 mM in both LnVBPOs, K_m for were 0.53 mM for LnVBPO1 and 0.35 mM for LnVBPO2, respectively. A derivative of putative precursor of laurencin (1), TMS-capped (3E, 6R, 7R)-laurediol (4) was subjected to LnVBPOs enzymatic bromination and gave similar MS/MS spectrum of deacetylaurencin (3). Since the compound 3 is considered to be the final precursor of 1, it is suggested that VBPO catalyzes bromination in Laurenciametabolites. We also cloned VBPOs from other Laurencia spp. which produce brominated sesquiterpenoids (L. okamurae) and triterpenoids (L. saitoi). We will discuss in vitro enzymatic bromination of these two LaurenciaVBPOs.
• Draft genome sequence of the biofilm-producing Bacillus subtilis strain B-1, isolated from an oil field

  S. Kesel, F. Moormann, I. G�mperlein, A. Mader, M. Morikawa, O. Lieleg, M. Opitz

  Genome Announcements 2 (6) e01163-14  2169-8287 2014 [Refereed][Not invited]
   

  We report here the draft genome sequence of the Bacillus subtilis strain B-1, a strain known to form biofilms. The biofilm matrix mainly consists of the biopolymer γ-polyglutamate (γ-PGA). The sequence of the genome of this strain allows the study of specific genes involved in biofilm formation.
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• Production and Characterization of a Biosurfactant from Cyberlindnera samutprakarnensis JP52(T)

  Jamroonsri Poomtien, Jiraporn Thaniyavarn, Pairoh Pinphanichakarn, Sasitorn Jindamorakot, Masaaki Morikawa

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 77 (12) 2362 - 2370 0916-8451 2013/12 [Refereed][Not invited]
   

  Cyberlindnera samutprakarnensis JP52(T), isolated from cosmetic industrial wastes in Thailand, was found to be an efficient biosurfactant-producing yeast when cultured in a medium containing (2% (w/v) glucose and 2% (v/v) palm oil at 30 degrees C, 200 rpm for 7 d. The crude biosurfactant had the ability to reduce the surface tension from 55.7 to 30.9 mN/m at 25 degrees C with a critical micelle concentration (CMC) of 0.046%. Physicochemical analysis of the crude biosurfactant revealed that it had wide ranges of optimum pH and pH stability at 6-9 and 3-10 respectively. It was also thermostable and retained 80% activity even after heat treatment, and it tolerated NaCI at 1.0-10%. Furthermore, it effectively emulsified various vegetable oils with an E24 value of over 80%. A partially purified biosurfactant fraction was analyzed for its structure by MALDI-TOF MS and NMR. This revealed that the biosurfactant mainly contained sophorolipids in C18-(MW 574) and C16-diaceltylated (MW 662) forms.
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• Isolation and Characterization of a Thermotolerant Ammonia-Oxidizing Bacterium Nitrosomonas sp JPCCT2 from a Thermal Power Station

  Yoshikane Itoh, Keiko Sakagami, Yoshihito Uchino, Chanita Boonmak, Tetsuro Oriyama, Fuyumi Tojo, Mitsufumi Matsumoto, Masaaki Morikawa

  MICROBES AND ENVIRONMENTS 28 (4) 432 - 435 1342-6311 2013/12 [Refereed][Not invited]
   

  A thermotolerant ammonia-oxidizing bacterium strain JPCCT2 was isolated from activated sludge in a thermal power station. Cells of JPCCT2 are short non-motile rods or ellipsoidal. Molecular phylogenetic analysis of 16S rRNA gene sequences demonstrated that JPCCT2 belongs to the genus Nitrosomonas with the highest similarity to Nitrosomonas nitrosa Nm90 (100%), Nitrosomonas sp. Nm148 (99.7%), and Nitrosomonas communis Nm2 (97.7%). However, G+C content of JPCCT2 DNA was 49.1 mol% and clearly different from N. nitrosa Nm90, 47.9%. JPCCT2 was capable of growing at temperatures up to 48 degrees C, while N. nitrosa Nm90 and N. communis Nm2 could not grow at 42 degrees C. Moreover, JPCCT2 grew similarly at concentrations of carbonate 0 and 5 gL(-1). This is the first report that Nitrosomonas bacterium is capable of growing at temperatures higher than 37 degrees C.
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• Draft genome sequence of Geobacillus thermoleovorans strain B23

  Chanita Boonmak, Yasunori Takahasi, Masaaki Morikawa

  Genome Announcements 1 (6) e00944-13  2169-8287 2013 [Refereed][Not invited]
   

  Here, we report the draft genome sequence of Geobacillus thermoleovorans strain B23, which was isolated from a deep subterranean petroleum reservoir in Japan. An array of genes related to unique long-chain alkane degradation pathways in G. thermoleovorans B23 has been identified by whole-genome analyses of this strain.
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• Sustainable biodegradation of phenolic endocrine-disrupting chemicals by Phragmites australis-rhizosphere bacteria association

  T. Toyama, T. Ojima, Y. Tanaka, K. Mori, M. Morikawa

  WATER SCIENCE AND TECHNOLOGY 68 (3) 522 - 529 0273-1223 2013 [Refereed][Not invited]
   

  The efficacy of two rhizobacteria (Sphingobium fuliginis TIK1 and Sphingobium sp. IT4) of Phragmites australis for the sustainable treatment of water polluted with phenolic endocrine-disrupting chemicals (EDCs) was investigated. Strains TIK1 and IT4 have recently been isolated from Phragmites rhizosphere and shown to degrade various 4-alkylphenols-TIK1 via phenolic ring hydroxylation and meta-cleavage and IT4 via ipso-hydroxylation. The two strains also degraded bisphenol A (BPA), bisphenol B, bisphenol E, bisphenol F, bisphenol P and bisphenol S (BPS). Thus, strains TIK1 and IT4 have wide degradation spectra for phenolic EDCs. The two strains utilized Phragmites root extracts as a sole carbon source and sustainably colonized Phragmites roots, where they degraded phenolic EDCs. In sequencing batch reactor experiments using Phragmites in association with TIK1 or IT4, both associations repeatedly removed phenolic EDCs from polluted secondary effluent water (BPA, BPS, 4-tert-butylphenol, 4-tert-octylphenol and 4-nonylphenol) from polluted secondary effluent water. The results suggest that hydroponic systems using Phragmites-TIK and Phragmites-IT4 associations would be useful for sustainable treatment of polluted waters containing various phenolic EDCs.
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• バイオフィルムをしらべてみよう

  森川 正章

  生物工学会誌 90 246 - 250 2012/05 [Not refereed][Invited]
  
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• Efficacy of forming biofilms by naphthalene degrading Pseudomonas stutzeri T102 toward bioremediation technology and its molecular mechanisms

  Kohei Shimada, Yoshikane Itoh, Kenji Washio, Masaaki Morikawa

  CHEMOSPHERE 87 (3) 226 - 233 0045-6535 2012/04 [Refereed][Not invited]
   

  In natural environments, bacteria often exist in close association with surfaces and interfaces. There they form "biofilms", multicellular aggregates held together by an extracellular matrix. The biofilms confer on the constituent cells high resistance to environmental stresses and diverse microenvironments that help generate cellular heterogeneity. Here we report on the ability of Pseudomonas stutzeri T102 biofilm-associated cells, as compared with that of planktonic cells, to degrade naphthalene and survive in petroleum-contaminated soils. In liquid culture system. T102 biofilm-associated cells did not degrade naphthalene during initial hours of incubation but then degraded it faster than planktonic cells, which degraded naphthalene at a nearly constant rate. This delayed but high degradation activity of the biofilms could be attributed to super-activated cells that were detached from the biofilms. When the fitness of T102 biofilm-associated cells was tested in natural petroleum-contaminated soils, they were capable of surviving for 10 wk; by then T102 planktonic cells were mostly extinct. Naphthalene degradation activity in the soils that had been inoculated with T102 biofilms was indeed higher than that observed in soils inoculated with T102 planktonic cells. These results suggest that inoculation of contaminated soils with P. stutzeri T102 biofilms should enable bioaugmentation to be a more durable and effective bioremediation technology than inoculation with planktonic cells. (C) 2012 Elsevier Ltd. All rights reserved.
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• Unique Microbiological Characteristics of Pseudoalteromonas spp. that Form Biofilms in the Sea Water Environment : Their Possible Contribution to Water Purification and Fish Farming

  IIJIMA Saori, OKAHARA Ryota, WASHIO Kenji, MORIKAWA Masaaki

  Technical Report on Salt Science The Society of Sea Water Science, Japan 66 (4) 186 - 190 0369-4550 2012 [Not refereed][Not invited]
  
• A Truncated Form of SpoT, Including the ACT Domain, Inhibits the Production of Cyclic Lipopeptide Arthrofactin, and Is Associated with Moderate Elevation of Guanosine 3 ',5 '-Bispyrophosphate Level in Pseudomonas sp MIS38

  Kenji Washio, Siew Ping Lim, Niran Roongsawang, Masaaki Morikawa

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 75 (10) 1880 - 1888 0916-8451 2011/10 [Refereed][Not invited]
   

  Arthrofactin is a biosurfactant produced by Pseudomonas sp. MIS38. We have reported that transposon insertion into spoT (spoT::Tn5) causes moderate accumulation of guanosine 3',5'-bispyrophosphate (ppGpp) and abrogates arthrofactin production. To analyze the linkage of SpoT function and ablation of arthrofactin production, we examined the spoT::Tn5 mutation. The results showed that spoT::Tn5 is not a null mutation, but encodes separate segments of SpoT. Deletion of the 3' region of spoT increased the level of arthrofactin production, suggesting that the C-terminal region of SpoT plays a suppressive role. We evaluated the expression of a distinct segment of SpoT. Forced expression of the C-terminal region that contains the ACT domain resulted in the accumulation of ppGpp and abrogated arthrofactin production. Expression of the C-terminal segment also reduced MIS38 swarming and resulted in extensive biofilm formation, which constitutes the phenocopy of the spoT::Tn5 mutant.
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• Diversity of Nonribosomal Peptide Synthetases Involved in the Biosynthesis of Lipopeptide Biosurfactants

  Niran Roongsawang, Kenji Washio, Masaaki Morikawa

  INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES 12 (1) 141 - 172 1422-0067 2011/01 [Refereed][Not invited]
   

  Lipopeptide biosurfactants (LPBSs) consist of a hydrophobic fatty acid portion linked to a hydrophilic peptide chain in the molecule. With their complex and diverse structures, LPBSs exhibit various biological activities including surface activity as well as anti-cellular and anti-enzymatic activities. LPBSs are also involved in multi-cellular behaviors such as swarming motility and biofilm formation. Among the bacterial genera, Bacillus (Gram-positive) and Pseudomonas (Gram-negative) have received the most attention because they produce a wide range of effective LPBSs that are potentially useful for agricultural, chemical, food, and pharmaceutical industries. The biosynthetic mechanisms and gene regulation systems of LPBSs have been extensively analyzed over the last decade. LPBSs are generally synthesized in a ribosome-independent manner with megaenzymes called nonribosomal peptide synthetases (NRPSs). Production of active-form NRPSs requires not only transcriptional induction and translation but also post-translational modification and assemblage. The accumulated knowledge reveals the versatility and evolutionary lineage of the NRPSs system. This review provides an overview of the structural and functional diversity of LPBSs and their different biosynthetic mechanisms in Bacillus and Pseudomonas, including both typical and unique systems. Finally, successful genetic engineering of NRPSs for creating novel lipopeptides is also discussed.
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• Analyses of three dominant membrane proteins from anammox planctomycete Candidatus ‘Brocadia sinica’

  Tojo F, Itoh Y, Okabe S, Morikawa M

  J. Environ. Biotechnol. 環境バイオテクノロジー学会 11 (1-2) 77 - 81 1347-1856 2011 [Refereed][Not invited]
  
• Dioxygen activation responsible for oxidation of aliphatic and aromatic hydrocarbon compounds: current state and variants

  Masaaki Morikawa

  APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 87 (5) 1595 - 1603 0175-7598 2010/08 [Refereed][Not invited]
   

  The most significant aspect in microbial metabolisms, especially those of bacteria and archaea, is their marvelously wide acceptability of substrate electron donors and acceptors. This feature makes them to be attractive catalysts for environmental biotechnology in terms of degradation of harmful recalcitrant compounds, including hydrocarbons. Transformation of highly reduced and inert hydrocarbon compounds is with no doubt a challenging biochemical reaction for a single enzyme. However, several multi-component enzyme systems enable microorganisms to utilize hydrocarbons as carbon and energy (electron) sources. Initial biological attack to hydrocarbons is, in most cases, the hydroxylation that requires molecular dioxygen as a co-substrate. Dioxygen also contributes to the ring cleavage reaction of homo- and hetero-cyclic aromatic hydrocarbons. Although the molecular dioxygen is omnipresent and highly soluble in water, activation and splitting this triplet ground-state molecule to wed with difficult hydrocarbons need special devices. Non-heme iron, heme iron, or flavin nucleotide was designated as a major hidden dagger for this purpose.
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• Sustainable Biodegradation of Phenol by Acinetobacter calcoaceticus P23 Isolated from the Rhizosphere of Duckweed Lemna aoukikusa

  Fumiko Yamaga, Kenji Washio, Masaaki Morikawa

  ENVIRONMENTAL SCIENCE & TECHNOLOGY 44 (16) 6470 - 6474 0013-936X 2010/08 [Refereed][Not invited]
   

  Phenol-degrading bacteria were isolated from the rhizosphere of duckweed (Lemna aoukikusa) using an enrichment culture method. One of the isolates, P23, exhibited an excellent ability to degrade phenol and attach to a solid surface under laboratory conditions. Phylogenetic analysis revealed that P23 belongs to the genera Acinetobacter and has the highest similarity to Acinetobacter calcoaceticus. P23 rapidly colonized on the surface of sterilized duckweed roots and formed biofilms, indicating that the conditions provided by the root system of duckweed are favorable to P23. A long-term performance test (160 h) showed that continuous removal of phenol can be attributed to the beneficial symbiotic interaction between duckweed and P23 P23 is the first growth-promoting bacterium identified from Lemna aoukikusa. The results in this study suggest the potential usefulness of dominating a particular bacterium in the rhizosphere of duckweeds to achieve efficient and sustainable bioremediation of polluted water
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• Identification and Characterization of the Genes Responsible for the Production of the Cyclic Lipopeptide Arthrofactin by Pseudomonas sp MIS38

  Kenji Washio, Siew Ping Lim, Niran Roongsawang, Masaaki Morikawa

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 74 (5) 992 - 999 0916-8451 2010/05 [Refereed][Not invited]
   

  Pseudomonas sp. MIS38 produces an effective biosurfactant named arthrofactin, which is a cyclic lipopeptide synthesized by a mega complex composed of three nonribosomal peptide synthetases. In order to gain insight into the control mechanism of arthrofactin production, a Tn5 mutant library was constructed and screened for arthrofactin-deficient mutants. Along with a number of mutations that occurred in the arthrofactin synthetase operon, three other mutants harbored distinct Tn5 insertions in the genes encoding SyrF-like protein (arfF), heat shock protein (htpG), and (p)ppGpp synthetase/hydrolase (spoT). Epistasis analyses revealed that spoT functions early in the arthrofactin production pathway. We also found that spoT affects MIS38 swarming, biofilm formation, and the cell morphology.
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• The Role of Urease Activity on Biofilm Formation by Staphylococcus sp T-02 Isolated from the Toilet Bowl

  Kaihei Oki, Kenji Washio, Daigo Matsui, Shinichi Kato, Yoshihiko Hirata, Masaaki Morikawa

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 74 (3) 583 - 589 0916-8451 2010/03 [Refereed][Not invited]
   

  Urolith, which consists of dirty yellow-colored attachments on the toilet bowl, is associated with a variety of odorous chemicals, including ammonia, and causes disadvantages in daily life. Although largely it is derived from microorganisms, little is known about the microbial processes underlying the formation of urolith. In order to gain insight into the types and the activities of microorganisms present in urolith, culturable bacteria were isolated, identified, and physiologically characterized. One of the isolates exhibited higher ability to produce ammonia when it was grown in artificial urine medium. Phylogenetic and physiological analyses indicated that this strain (T-02) belonged to a new group of Staphylococcus species, showing combined phenotypes as between S. lentus and S. xylosus. T-02 exhibited high urease activity and was capable of growing in the urinary condition by forming robust biofilms. The results of this study indicate that T-02 has successfully adapted itself to the environment of urolith.
• Feasibility of bioremediation technologies for managing oil spills caused by the development of the Sakhalin oil fields-Review-

  Sakaguchi Naofumi, Bin Haji Mohd Taha Ahmad Iskandar, Maki Hideaki, Hamada Seiichi, Morikawa Masaaki, Okuyama Hidetoshi

  生物工学会誌 日本生物工学会 88 (4) 150 - 157 0919-3758 2010 [Refereed][Not invited]
   

  The oil fields at Sakhalin Island, Russia, reached a high level of operation in 2008. The Japanese island of Hokkaido is located to the south of Sakhalin, and is at risk of oil spills following explosions at the Sakhalin oil fields and from accidents involving pipelines and tankers at the fields and in the surrounding seas. Physical, chemical, and biological treatments are indispensable in cleaning up any contaminated regions, such as seas and seashores. In this review, we describe various types of accidents, particularly those involving tanker oil spills around the world and the bioremediation (BR) treatment employed. We emphasize the usefulness of BR in colder regions, such as Hokkaido. Pioneering on-site biostimulation (BS) testing has been performed on the Hokkaido coast with the aim of dealing with oil contamination caused by oil spills at Sakhalin. In the BS process, appropriate nutrients other than carbon sources are supplied to contaminated soils or waters to activate native degraders of the contaminants. In another bioaugmentation (BA) process, isolated microorganisms and their mixtures can be used as degraders of xenobiotics. We proposed and tested a new bioremediation technology, autochthonous bioaugmentation (ABA), in which contaminant-degrading microorganisms indigenous to the contaminated site or predicted contamination site are isolated and used. The feasibility of applying ABA to oil spills caused by the development of the Sakhalin oil fields is discussed.
• Gene cloning and characterization of an aldehyde dehydrogenase from long-chain alkane-degrading Geobacillus thermoleovorans B23

  Tomohisa Kato, Asuka Miyanaga, Shigenori Kanaya, Masaaki Morikawa

  EXTREMOPHILES 14 (1) 33 - 39 1431-0651 2010/01 [Refereed][Not invited]
   

  Geobacillus thermoleovorans B23 is capable of degrading long-chain alkanes at 70A degrees C. Bt-aldh, an aldehyde dehydrogenase gene in B23, was located in the upstream region of p21 whose expression level was dramatically increased when alkane degradation was started (Kato et al. 2009, BMC Microbiol 9:60). Like p21, transcription level of Bt-aldh was also increased upon alkane degradation. Bt-Aldh (497 aa, MW = 53,886) was overproduced in Escherichia coli, purified, and characterized biochemically. Bt-Aldh acted as an octamer, required NAD(+) as a coenzyme, and showed high activity against aliphatic long-chain aldehydes such as tetradecanal. The optimum condition for activity was 50-55A degrees C and pH 10.0. The activity was elevated to two- to threefold in the presence of 2 mM Ba2+, Ca2+, or Sr2+, while Mg2+ and Zn2+ inhibited the enzyme activity. Bt-Aldh represents thermophilic aldehyde dehydrogenases responsible for degradation of long-chain alkanes.
• Autochthonous bioaugmentation and its possible application to oil spills

  Reia Hosokawa, Motonori Nagai, Masaaki Morikawa, Hidetoshi Okuyama

  WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY 25 (9) 1519 - 1528 0959-3993 2009/09 [Refereed][Not invited]
   

  Bioaugmentation for oil spills is a much more promising technique than is biostimulation. However, the effectiveness of bioaugmentation is variable, because the survival and the xenobiotic-degrading ability of introduced microorganisms are highly dependent on environmental conditions. As an alternative, autochthonous bioaugmentation (ABA) is proposed to overcome these difficulties. The ABA method is like a ready-made bioaugmentation technology. In ABA, microorganisms indigenous to the contaminated site or predicted contamination site that are well-characterized and potentially capable of degrading oils are used, and these microorganisms should be enriched under conditions where bioaugmentation will be conducted. It is possible to obtain information in advance on the chemical and physical characteristics of potential oil spill sites and of oils that might be spilled. The application of ABA in the coastal areas of Hokkaido Prefecture, Japan, is considered here, because Hokkaido is located south of Sakhalin Island, Russia, where development of oil fields is in progress. If oil spills in this region were well characterized in advance, ABA could be a feasible technology in the near future.
• Flexible exportation mechanisms of arthrofactin in Pseudomonas sp MIS38

  S. P. Lim, N. Roongsawang, K. Washio, M. Morikawa

  JOURNAL OF APPLIED MICROBIOLOGY 107 (1) 157 - 166 1364-5072 2009/07 [Refereed][Not invited]
   

  To obtain further insights into transportation mechanisms of a most effective biosurfactant, arthrofactin in Pseudomonas sp. MIS38. A cluster genes arfA/B/C encodes an arthrofactin synthetase complex (ArfA/B/C). Downstream of the arfA/B/C lie genes encoding a putative periplasmic protein (ArfD, 362 aa) and a putative ATP-binding cassette transporter (ArfE, 651 aa), namely arfD and arfE, respectively. The arfA/B/C, arfD, and arfE form an operon suggesting their functional connection. Gene knockout mutants ArfD:Km, ArfE:Km, ArfD:Tc/ArfE:Km, and gene overexpression strains MIS38(pME6032_arfD/E) and ArfE:Km(pME6032_arfD/E) were prepared and analysed for arthrofactin production profiles. It was found that the production levels of arthrofactin were temporally reduced in the mutants or increased in the gene overexpression strains, but they eventually became similar level to that of MIS38. Addition of ABC transporter inhibitors, glibenclamide and sodium ortho-vanadate dramatically reduced the production levels of arthrofactin. This excludes a possibility that arthrofactin is exported by diffusion with the aid of its own high surfactant activity. ArfD/E is not an exclusive but a primary exporter of arthrofactin during early growth stage. Reduction in the arthrofactin productivity of arfD and arfE knockout mutants was eventually rescued by another ABC transporter system. Effects of arfD and arfE overexpression were evident only for 1-day cultivation. Multiple ATP dependent active transporter systems are responsible for the production of arthrofactin. Pseudomonas bacteria are characterized to be endued with multiple exporter and efflux systems for secondary metabolites including antibiotics, plant toxins, and biosurfactants. The present work demonstrates exceptionally flexible and highly controlled transportation mechanisms of a most effective lipopeptide biosurfactant, arthrofactin in Pseudomonas sp. MIS38. Because lipopeptide biosurfactants are known to enhance efficacy of bioactive compounds and arfA/B/C/D/E orthologous genes are also found in plant pathogenic P. fluorescens and P. syringae strains, the knowledge would also contribute to develop a technology controlling plant diseases.
• Biofilm formation and proteolytic activities of Pseudoalteromonas bacteria that were isolated from fish farm sediments

  Saori Iijima, Kenji Washio, Ryota Okahara, Masaaki Morikawa

  MICROBIAL BIOTECHNOLOGY 2 (3) 361 - 369 1751-7907 2009/05 [Refereed][Not invited]
   

  In order to save natural resources and supply good fishes, it is important to improve fish-farming techniques. The survival rate of fish fry appears to become higher when powders of foraminifer limestone are submerged at the bottom of fish-farming fields, where bacterial biofilms often grow. The observations suggest that forming biofilms can benefit to keep health status of breeding fishes. We employed culture-based methods for the identification and characterization of biofilm-forming bacteria and assessed the application of their properties for fish farming. Fifteen bacterial strains were isolated from the biofilm samples collected from fish farm sediments. The 16S rRNA gene sequences indicated that these bacteria belonged to the genera, Pseudoalteromonas (seven strains), Vibrio (seven strains) and Halomonas (one strain). It was found that Pseudoalteromonas strains generally formed robust biofilms in a laboratory condition and produced extracellular proteases in a biofilm-dependent manner. The results suggest that Pseudoalteromonas bacteria, living in the biofilm community, contribute in part to remove excess proteineous matters from the sediment sludge of fish farms.
• Alkane inducible proteins in Geobacillus thermoleovorans B23

  Tomohisa Kato, Asuka Miyanaga, Shigenori Kanaya, Masaaki Morikawa

  BMC MICROBIOLOGY 9 60  1471-2180 2009/03 [Refereed][Not invited]
   

  Background: Initial step of beta-oxidation is catalyzed by acyl-CoA dehydrogenase in prokaryotes and mitochondria, while acyl-CoA oxidase primarily functions in the peroxisomes of eukaryotes. Oxidase reaction accompanies emission of toxic by-product reactive oxygen molecules including superoxide anion, and superoxide dismutase and catalase activities are essential to detoxify them in the peroxisomes. Although there is an argument about whether primitive life was born and evolved under high temperature conditions, thermophilic archaea apparently share living systems with both bacteria and eukaryotes. We hypothesized that alkane degradation pathways in thermophilic microorganisms could be premature and useful to understand their evolution. Results: An extremely thermophilic and alkane degrading Geobacillus thermoleovorans B23 was previously isolated from a deep subsurface oil reservoir in Japan. In the present study, we identified novel membrane proteins (P16, P21) and superoxide dismutase (P24) whose production levels were significantly increased upon alkane degradation. Unlike other bacteria acyl-CoA oxidase and catalase activities were also increased in strain B23 by addition of alkane. Conclusion: We first suggested that peroxisomal beta-oxidation system exists in bacteria. This eukaryotic-type alkane degradation pathway in thermophilic bacterial cells might be a vestige of primitive living cell systems that had evolved into eukaryotes.
• Development of bioremediation by utilizing biofilms.

  MORIKAWA, Masaaki

  IFO Res. Commun. 23 99 - 115 2009 [Refereed][Not invited]
  
• Identification of alkane hydroxylase genes in Rhodococcus sp strain TMP2 that degrades a branched alkane

  Daisuke Takei, Kenji Washio, Masaaki Morikawa

  BIOTECHNOLOGY LETTERS 30 (8) 1447 - 1452 0141-5492 2008/08 [Refereed][Not invited]
   

  Rhodococcus sp. TMP2 is an alkane-degrading strain that can grow with a branched alkane as a sole carbon source. TMP2 degrades considerable amounts of pristane at 20 degrees C but not at 30 degrees C. In order to gain insights into microbial alkane degradation, we characterized one of the key enzymes for alkane degradation. TMP2 contains at least five genes for membrane-bound, non-heme iron, alkane hydroxylase, known as AlkB (alkB1-5). Phylogenetical analysis using bacterial alkB genes indicates that TMP2 is a close relative of the alkane-degrading bacteria, such as Rhodococcus erythropolis NRRL B-16531 and Q15. RT-PCR analysis showed that expressions of the genes for AlkB1 and AlkB2 were apparently induced by the addition of pristane at a low temperature. The results suggest that TMP2 recruits certain alkane hydroxylase systems to utilize a branched alkane under low temperature conditions.
• Production of sophorolipid biosurfactant by Pichia anomala

  Jiraporn Thaniyavarn, Tanusta Chianguthai, Polakit Sangvanich, Niran Roongsawang, Keji Washio, Masaaki Morikawa, Suthep Thaniyavarn

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 72 (8) 2061 - 2068 0916-8451 2008/08 [Refereed][Not invited]
   

  Biosurfactant production by Pichia anomala PY1, a thermotorelant strain isolated from fermented food, was examined as grown in media containing various carbon and nitrogen sources. The optimal conditions for biosurfactant production included 4% soybean oil as carbon source at pH 5.5 at 30 degrees C for 7d. Under these conditions, the surface tension of the medium decreased to 28mN/m with oil displacement measured at 69.43 cm(2). Comparative studies of biosurfactant production in media containing glucose or soybean oil were performed. The biosurfactants obtained were isolated and purified by chromatographic methods. The molecular weights of samples were further investigated by mass spectrometry. In medium containing glucose, biosurfactants of molecular weights of 675, 691, and 707 were obtained, while those isolated from medium containing soybean oil were of molecular weights of 658, 675, and 691. These results reveal that sophorolipid compounds containing fatty acids of C20 and C18:1 were produced from both media.
• A turbine oil-degrading bacterial consortium from soils of oil fields and its characteristics

  Hitoshi Ito, Reia Hosokawa, Masaaki Morikawa, Hidetoshi Okuyama

  INTERNATIONAL BIODETERIORATION & BIODEGRADATION 61 (3) 223 - 232 0964-8305 2008/04 [Refereed][Not invited]
   

  A microbial consortium capable of degrading turbine oil (TuO), which consisted mainly of recalcitrant cycloalkanes and isoalkanes, was obtained from a soil sample collected from oil fields using repeated enrichment. When this consortium, named Atsuta A, was cultured in minimal salts medium containing 0.5% (w/v) TuO, it degraded 90% of TuO at 30 degrees C and pH 7 over 5 days. Although nine bacterial strains were isolated from the Atstita A consortium, TuO degradation by the individual isolates and by a mixture of them was negligible. The community structure of the consortium, which was investigated by PCR-denaturing gradient gel electrophoresis (DGGE) targeting 16S rRNA genes, changed significantly during the degradation of TuO. Four major bands (F, K, N and T) out of at least 23 DGGE bands significantly increased in intensity over time during incubation. The DGGE bands F, K and N corresponded to those of previously isolated species. However, DGGE band T did not correspond to any isolated strain. The 16S rRNA gene sequence collected from band T was 98% homologous to that of an unculturable strain belonging to the gamma-Proteobacteria. The degradation of TuO in the consortium may occur by cooperation between the unculturable species corresponding to band T and other strains in the consortium, including species corresponding to bands F, K and N. (C) 2007 Elsevier Ltd. All rights reserved.
• Duckweed-Rhizobacteria Symbiosis that Develops Sustainable Remediation Technology

  YAMAGA Fumiko, WASHIO Kenji, MORIKAWA Masaaki

  KAGAKU TO SEIBUTSU Japan Society for Bioscience, Biotechnology, and Agrochemistry 46 (10) 682 - 688 0453-073X 2008 [Refereed][Not invited]
   

  微生物活性を利用した環境浄化法は，地球への負荷が少ない技術として広く認知されている．しかし，実用的技術としては，有用微生物の現場での定着率の低さによるコスト高や浄化後に危惧される微生物の異常増殖や生態系の攪乱などの問題が常に指摘されてきた．近年，この問題を解決する一つの方向性として根圏微生物の活用が注目されている．植物の光合成作用と微生物の汚染物質分解活性をリンクさせるこの手法の一例を紹介するとともに，今後の可能性を展望する．
• Cloning of hyaluronan synthase (sz-hos) gene from Streptococcus zooepidemicus in Escherichia coli

  Boonsri Jongsareejit, Amaret Bhumiratana, Masaaki Morikawa, Shigerri Kanaya

  ScienceAsia 33 (4) 389 - 395 1513-1874 2007/12 [Refereed][Not invited]
   

  A 546-bp fragment of the sz-hasA gene encoding hyaluronan synthase (szHAS) from Streptococcus zooepidemicus (group C Streptococcus, GCS) was amplified by PCR with oligonucleotidesdesigned based on the conserved amino acid sequences of HASs from other organisms as primers. The entire sz-hasA gene was identified and cloned by Southern and colony hybridizations using this 546-bp fragment as a probe. Determination of the nucleotide sequence indicated that this gene encoded a protein with 417 amino acid residues (calculated molecular mass, 47.77 kDa). The amino acid sequence of szHAS was 74.2% identical to that of HAS from Strep. equisimilis. The overexpression of sz-hasA in Escherichia coli was detected by SDS-PAGE and confirmed by LC/MS-MS. To examine whether E. coli cells acquire an ability to synthesize hyaluronic acid (HA) upon transformation with plasmids bearing the sz-hasA gene, theplasmids pHAS-1 and pHAS-2, in which sz-hasA and sz-hasA with rare codon modifications at the 5′-terminus were ligated into the Ndel-BamHI sites of pET-28a, respectively, were constructed and used to transform E. coli BL21 (DE3), E. coli BL21 (codon+), and E. coli HMS 174 (DE3) plysS. Cultivation of these transformants, followed by induction for gene expression, revealed that the E. coli BL21 (codon+) transformants with pHAS-1 and E. coli HMS 174 (DE3) plysS transformants with pHAS-2 produced HA after 4 hr induction time at the maximum yield 16 μg/ml and 32.5 μg/ml, respectively. These are higher than the background levels in control bacteria, which were 5 μg/ml and 21.3 μg/ml, respectively.
• Functional analysis of a pyoverdine synthetase from Pseudomonas sp MIS38

  Slew Ping Lim, Niran Roongsawang, Kenji Washio, Masaaki Morikawa

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 71 (8) 2002 - 2009 0916-8451 2007/08 [Refereed][Not invited]
   

  Fluorescent Pseudomonas sp. MIS38 produces a cyclic lipopeptide, arthr4actin. Arthrofactin is synthesized by a unique nonribosomal peptide synthetase (NRPS) with dual C/E-domains. In this study, another class of cyclic peptide, pyoverdine, was isolated from MIS38, viz., Pvd38. The main fraction of Pvd38 had an m/z value of 1,064.57 and contained Ala, Glu, Gly, (OHOrn), Ser, and Thr at a ratio of 2:1:1:(1):1:1 in the peptide part, suggesting a new structure compound. A gene encoding NRPS for the chromophore part of Pvd38 was identified, and we found that it contained a conventional E-domain. Gene disruption completely impaired the production of Pvd38, demonstrating that the synthetase is functional. This observation allows us to conclude that different NRPS systems with dual C/Edomains (in arthrofactin synthetase) and a conventional E-domain (in pyoverdine synthetase) are both functional in MIS38.
• In vivo characterization of tandem C-terminal thioesterase domains in arthrofactin synthetase

  Niran Roongsawang, Kenji Washio, Masaaki Morikawa

  CHEMBIOCHEM 8 (5) 501 - 512 1439-4227 2007/03 [Refereed][Not invited]
   

  Mocrocyclization of a peptide or a lipopeptide occurs at the last step of synthesis and is usually catalyzed by a single C-terminal thioesterase (Te) domain. Arthrofactin synthetase WO from Pseudomonas sp. MIS38 represents a novel type of nonribosomal peptide synthetase that contains unique tandem C-terminal Te domains, ArfC_Te1 and ArfC_Te2. In order to analyze their function in vivo, site-directed mutagenesis was introduced at the putotive active-site residues in ArfC_Te1 and ArfC_Te2. It was found that both Te domains were functional. Peaks corresponding to arthrofactin and its derivatives were absent in ArfC_Te1.S89A, ArfC_Te1.-S89T, and ArfC_Te1:E26G/F27A mutants, and the production of arthrofactin by ArfC_7e2:S92A, ArfC_7e2:S92A/D118A, and AnFC Delta 7e2 was reduced by 95% without an alteration of the cyclic lipoundecapeptide structure. These results suggest that Ser89 in ArfC_Te1 is essential for the completion of macrocyclization and the release of product. Glu26 and Phe27 residues are also part of the active site of ArfC_Te1. ArfC_7e2 might hove been added during the evolution of Arf in order to improve macrocyclization efficiency.
• バイオフィルムの功罪

  森川 正章

  食品衛生学雑誌 48 J389 - J396 2007 [Refereed][Not invited]
  
• Gentisate 1,2-dioxygenase from Xanthobacter polyaromaticivorans 127W

  Shin-ichi Hirano, Masaaki Morikawa, Kazufumi Takano, Tadayuki Imanaka, Shigenori Kanaya

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 71 (1) 192 - 199 0916-8451 2007/01 [Refereed][Not invited]
   

  A putative gentisate 1,2-dioxygenase was encoded in the dibenzothiophene degradation gene cluster (dbd) from Xanthobacter polyaromaticivorans 127W. The deduced amino acid sequence showed high sequence similarity with gentisate dioxygenases from Pseudomonas alealigenes (AAD49427, 65% identical), Bradyrhizobium japonicum (NP766750, 64%), and P. aeruginosa (ZP_00135722, 54%), and moderate similarity with 1-hydroxy-2-naphthoate dioxygenase from Nocardioides sp. KP7 (BAA31235, 33%) and salicylate dioxygenase from Pseudaminobacter salicylatoxidans (AAQ91293, 33%). The enzyme, GDOxp, was heterologously produced in Escherichia coli and purified to homogeneity. GDOxp formed a tetramer and exhibited high dioxygenase activity against 1,4-dihydroxy 2-naphthoate as well as gentisate, suggesting unusually broad substrate specificity. GDOxp easily released ferrous ion under unfavorable temperature and pH conditions to become an inactive monomer protein. An inactive monomer protein can reconstitute a tetramer structure and restore enzyme activity in a cooperative manner upon the addition of ferrous ion. Chymotryptic digestion and protein truncation experiments suggested that the N-terminal region is important for the tetramerization of GDOxp.
• Common mechanisms regulating expression of rice aleurone genes that contribute to the primary response for gibberellin

  Kenji Washio, Masaaki Morikawa

  BIOCHIMICA ET BIOPHYSICA ACTA-GENE STRUCTURE AND EXPRESSION 1759 (10) 478 - 490 0167-4781 2006/10 [Refereed][Not invited]
   

  During germination of cereal seeds, aleurone cells respond to gibberellins (GA) by synthesizing and secreting hydrolytic enzymes that mobilize the reserved nutrients. It has been shown that products of early GA response genes, like a transcription factor GAMyb, act as key molecules leading to this regulation. Pivotal roles of GAMyb on expression of hydrolase genes have been well documented, whereas regulation of GAMyb expression itself remains obscure. In order to understand virtual mechanisms of the GA-mediated expression of genes, it is important to know how GA control expression of early GA response genes. Using an aleurone transient expression system of rice (Oryza sativa L.), we examined GA responsive domains of early GA response genes in the aleurone, such as GAMyb and OsDof3. The upstream promoter could not confer GA response. Extensive analyses revealed the presence of enhancer-like activities in a large first intron. In Arabidopsis, intron enhancers have been identified in MADS-box homeotic genes, AGAMOUS (AG) and FLOWERING LOCUS C (FLC), in which large introns should not only confer proper gene expressions, but also associate with gene silencing by covalent modifications of both DNA and historic. These evidences prompt us to assign that chromatin-based control might be important for initial GA action. Based on this assumption, we have identified DNA methylation of the GAMyb locus in germinated rice seeds. (c) 2006 Elsevier B.V All rights reserved.
• Biofilm formation by a Bacillus subtilis strain that produces gamma-polyglutamate

  Masaaki Morikawa, Shinji Kagihiro, Mitsuru Haruki, Kazufumi Takano, Steve Branda, Roberto Kolter, Shigenori Kanaya

  MICROBIOLOGY-SGM Society for General Microbiology 152 (9) 2801 - 2807 1350-0872 2006/09 [Refereed][Not invited]
   

  The extracellular matrix produced by Bacillus subtilis B-1, an environmental strain that forms robust floating biofilms, was purified, and determined to be composed predominantly of gamma-polyglutamate (gamma-PGA), with a molecular mass of over 1000 kDa. Both biofilm formation and gamma-PGA production by B. subtilis B-1 increased with increasing Mn2+ or glycerol concentration. gamma-PGA was produced in a growth-associated manner in standing culture, and floating biofilms were formed. However, gamma-PGA was produced in a non-growth-associated manner in shaking culture conditions. When B. subtilis B-1 was grown in a microaerated culture system, floating biofilm formation and gamma-PGA production were significantly retarded, suggesting that oxygen depletion is involved in the initial steps of floating biofilm formation in standing culture. Proteomic analysis of membrane proteins demonstrated that flagellin, oligopeptide permease and Vpr protease precursor were the major proteins produced by cells in a floating biofilm and a colony.
• Biosurfactant production by Pseudomonas aeruginosa A41 using palm oil as carbon source

  Jiraporn Thaniyavarn, Aree Chongchin, Nopparat Wanitsuksombut, Suthep Thaniyavarn, Pairoh Pinphanichakarn, Natthanant Leepipatpiboon, Masaaki Morikawa, Shigenori Kanaya

  JOURNAL OF GENERAL AND APPLIED MICROBIOLOGY 52 (4) 215 - 222 0022-1260 2006/08 [Refereed][Not invited]
   

  Biosurfactant production by Pseudomonas aeruginosa A41, a strain isolated from seawater in the gulf of Thailand, was examined when grown in defined medium containing 2% vegetable oil or fatty acid as a carbon source in the presence of vitamins, trace elements and,0.4% NH4NO3, at pH 7 and 30 C with 200 rpm-shaking for 7 days. The yield of biosurfactant steadily increased even after a stationary phase. Under such conditions the surface tension of the medium was lowered from 55-70 mN/m to 27.8-30 mN/m with every carbon source tested. However, types of carbon sources were found to affect biosurfactant yield. The yields of rhamnolipid biosurfactant were 6.58g/L, 2.91 g/L and 2.93g/L determined as rhamnose content when olive oil, palm oil and coconut oil, respectively, were used as a carbon source. Among them, biosurfactant obtained from palm oil was the best in lowering surface tension of the medium. Increase in biosurfactant activities in terms of oil displacement test and rhamnose, content were observed to be higher with shorter chain fatty acids than that of the longer chains (C12 > C14 > C16). In addition, we found that C18:2, highly unsaturated fatty acid, showed higher oil displacement activity and rhamnose content than that of C18:1. The optimal oil displacement activity was found at pH 7-9 and in the presence of 0.5-3% NaCl. The oil displacement activity was stable to temperatures up to 100 C for 15 h. Surface tension reduction activity was relatively stable at pH 2-12 and 0-5% of NaCl. Emusification activity tested with various types of hydrocarbons and vegetable oils showed similarity of up to 60% stability. The partially purified biosurfactant via TLC and silica gel column chromatography gave three main peaks on HPLC with mass spectra of 527, 272, and 661 m/z respectively, corresponding to sodium-monorhamnodecanoate, hydroxyhexadecanoic acid and an unknown compound, respectively.
• Ca2+-dependent maturation of subtilisin from a hyperthermophilic archaeon, Thermococcus kodakaraensis: the propeptide is a potent inhibitor of the mature domain but is not required for its folding

  M Pulido, K Saito, SI Tanaka, Y Koga, M Morikawa, K Takano, S Kanaya

  APPLIED AND ENVIRONMENTAL MICROBIOLOGY 72 (6) 4154 - 4162 0099-2240 2006/06 [Refereed][Not invited]
   

  Subtilisin from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 is a member of the subtilisin family. T. kodakaraensis subtilisin in a proform (T. kodakaraensis pro-subtilisin), as well as its propeptide (T. kodakaraensis propeptide) and mature domain (T. kodakaraensis mat-subtilisin), were independently overproduced in E. coli, purified, and biochemically characterized. T. kodakaraensis pro-subtilisin was inactive in the absence of Ca2+ but was activated upon autoprocessing and degradation of propeptide in the presence of Ca2+ at 80 degrees C. This maturation process was completed within 30 min at 80 degrees C but was bound at an intermediate stage, in which the propeptide is autoprocessed from the mature domain (T. kodakaraensis mat-subtilisin*) but forms an inactive complex with T. kodakaraensis mat-subtilisin*, at lower temperatures. At 80 degrees C, approximately 30% of T. kodakaraensis pro-subtilisin was autoprocessed into T. kodakaraensis propeptide and T. kodakaraensis mat-subtilisin*, and the other 70% was completely degraded to small fragments. Likewise, T. kodakaraensis mat-subtilisin was inactive in the absence of Ca2+ but was activated upon incubation with Ca2+ at 80 degrees C. The kinetic parameters and stability of the resultant activated protein were nearly identical to those of T. kodakaraensis mat-subtilisin*, indicating that T. kodakaraensis mat-subtilisin does not require T. kodakaraensis propeptide for folding. However, only similar to 5% of T. kodakaraensis mat-subtilisin was converted to an active form, and the other part was completely degraded to small fragments. T. kodakaraensis propeptide was shown to be a potent inhibitor of T. kodakaraensis mat-subtilisin* and noncompetitively inhibited its activity with a K-i of 25 +/- 3.0 nM at 20 degrees C. T. kodakaraensis propeptide may be required to prevent the degradation of the T. kodakaraensis mat-subtilisin molecules that are activated later by those that are activated earlier.
• Gene cloning and in vivo characterization of a dibenzothiophene dioxygenase from Xanthobacter polyaromaticivorans

  S Hirano, M Haruki, K Takano, T Imanaka, M Morikawa, S Kanaya

  APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 69 (6) 672 - 681 0175-7598 2006/02 [Refereed][Not invited]
   

  Xanthobacter polyaromaticivorans sp. nov. 127W is a bacterial strain that is capable of degrading a wide range of cyclic aromatic compounds such as dibenzothiophene, biphenyl, naphthalene, anthracene, and phenanthrene even under extremely low oxygen [dissolved oxygen (DO)<= 0.2 ppm] conditions (Hirano et al., Biosci Biotechnol Biochem 68:557-564, 2004). A major protein fraction carrying dibenzothiophene degradation activity was purified. Based on its partial amino acid sequences, dbdCa gene encoding alpha subunit terminal oxygenase (DbdCa) and its flanking region were cloned and sequenced. A phylogenetic analysis based on the amino acid sequence demonstrates that DbdCa is a member of a terminal oxygenase component of group IV ring-hydroxylating dioxygenases for biphenyls and monocyclic aromatic hydrocarbons, rather than group III dioxygenases for polycyclic aromatic hydrocarbons. Gene disruption in dbdCa abolished almost of the degradation activity against biphenyl, dibenzothiophene, and anthracene. The gene disruption also impaired degradation activity of the strain under extremely low oxygen conditions (DO <= 0.2 ppm). These results indicate that Dbd from 127W represents a group IV dioxygenase that is functional even under extremely low oxygen conditions.
• :Learning from the Microbial Activities which Exceed Central Dogma

  WASHIO Kenji, MORIKAWA Masaaki

  KAGAKU TO SEIBUTSU Japan Society for Bioscience, Biotechnology, and Agrochemistry 44 (2) 85 - 92 0453-073X 2006 [Refereed][Not invited]
  
• 枯草菌バイオフィルム形成遺伝子の探索

  森川正章, 河合良尚, 相原 悠, 鷲尾健司, 高野和文, 金谷茂則

  生物工学会誌 日本生物工学会 84 (12) 488 - 490 0919-3758 2006 [Refereed][Not invited]
  
• Crystal structure of TBP-interacting protein (Tk-TIP26) and implications for its inhibition mechanism of the interaction between TBP and TATA-DNA

  T Yamamoto, T Matsuda, T Inoue, H Matsumura, M Morikawa, S Kanaya, Y Kai

  PROTEIN SCIENCE 15 (1) 152 - 161 0961-8368 2006/01 [Refereed][Not invited]
   

  TATA-binding protein (TBP)-interacting protein from the hyperthermophilic archaeon Thermococcus kodakaraensis strain KODI (Tk-TIP26) is a possible transcription regulatory protein in Thermococcales. Here, we report the crystal structure of Tk-TIP26 determined at 2.3 angstrom resolution with multiple-wavelength anomalous dispersion (MAD) method. The overall structure of Tk-TIP26 consists of two domains. The N-terminal domain forms an alpha/beta structure, in which three alpha-helices enclose the central beta-sheet. The topology of this domain is similar to that of holliday junction resolvase Hjc from Pyrococcus furiosus. The C-terminal domain comprises three alpha-helices, six beta-strands, and two 3(10)-helices. In the dimer structure of Tk-TIP26, two molecules are related with the crystallographic twofold axis, and these molecules rigidly interact with each other via hydrogen bonds. The complex of Tk-TIP26/Tk-TBP is isolated and analyzed by SDS-PAGE and gel filtration column chromatography, resulting in a stoichiometric ratio of the interaction between Tk-TIP26 and Tk-TBP of 4:2, i.e., two dimer molecules of Tk-TIP26 formed a complex with one dimeric TBP. The electrostatic surfaces of Tk-TIP26 and TBP from Pyrocuccus woesei (PwTBP) allowed us to build a model of the Tk-TIP26/TBP complex, and to propose the inhibition mechanism where two dimer molecules of Tk-TIP26 bind to a dimeric TBP, preventing its binding to TATA-DNA.
• Beneficial biofilm formation by industrial bacteria Bacillus subtilis and related species

  M Morikawa

  JOURNAL OF BIOSCIENCE AND BIOENGINEERING 101 (1) 1 - 8 1389-1723 2006/01 [Refereed][Not invited]
   

  Biofilms are densely packed multicellular communities of microorganisms attached to a surface or interface. Bacteria seem to initiate biofilm formation in response to specific environmental cues, such as nutrient and oxygen availability. Biofilms undergo dynamic changes during their transition from free-living organisms to sessile biofilm cells, including the specific production of secondary metabolites and a significant increase in the resistivity to biological, chemical, and physical assaults. Bacillus subtilis is an industrially important bacterium exhibiting developmental stages. It forms rough biofilms at the air-liquid interface rather than on the surface of a solid phase in a liquid, due to the aerotaxis of the cells. Biofilm formation by B. subtilis and related species permits the control of infection caused by plant pathogens, the reduction of mild steel corrosion, and the exploration of novel compounds. Although it is obviously important to control harmful biofilm formation, the exploitation of beneficial biofilms formed by such industrial bacteria may lead to a new biotechnology.
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• Phylogenetic analysis of condensation domains in the nonribosomal peptide synthetases

  N Roongsawang, SP Lim, K Washio, K Takano, S Kanaya, M Morikawa

  FEMS MICROBIOLOGY LETTERS 252 (1) 143 - 151 0378-1097 2005/11 [Refereed][Not invited]
   

  Condensation (C) domains in the nonribosomal peptide synthetases are capable of catalyzing peptide bond formation between two consecutively bound various amino acids. C-domains coincide in frequency with the number of peptide bonds in the product peptide. In this study, a phylogenetic approach was used to investigate structural diversity of bacterial C-domains. Phylogenetic trees show that the C-domains are clustered into three functional groups according to the types of substrate donor molecules. They are L-peptidyl donors, D-peptidyl donors, and N-acyl donors. The fact that C-domain structure is not subject to optical configuration of amino acid acceptor molecules supports an idea that the conversion from L to D-form of incorporating amino acid acceptor occurs during or after peptide bond formation. L-peptidyl donors and D-peptidyl donors are suggested to separate before separating the lineage of Gram-positive and Gram-negative bacteria in the evolution process. (c) 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
• Gene cloning, overproduction, and characterization of thermolabile alkaline phosphatase from a psychrotrophic bacterium

  Y Suzuki, Y Mizutani, T Tsuji, N Ohtani, K Takano, M Haruki, M Morikawa, S Kanaya

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 69 (2) 364 - 373 0916-8451 2005/02 [Refereed][Not invited]
   

  The gene encoding alkaline phosphatase from the psychrotrophic bacterium Shewanella sp. SIB1 was cloned, sequenced, and overexpressed in Escherichia coli. The recombinant protein was purified and its enzymatic properties were compared with those of E. coli alkaline phosphatase (APase), which shows an amino acid sequence identity of 37%. The optimum temperature of SIB1 APase was 50degreesC, lower than that of E. coli APase by 30degreesC. The specific activity of SIB1 APase at 50degreesC was 3.1 fold higher than that of E. coli APase at 80degreesC. SIB1 APase lost activity with a half-life of 3.9min at 70degreesC, whereas E. coli APase lost activity with a half-life of >6h even at 80degreesC. Thus SIB1 APase is-well adapted to low temperatures. Comparison of the amino acid sequences of SIB1 and E. coli APases suggests that decreases in electrostatic interactions and number of disulfide bonds are responsible for the cold-adaptation of SIB1 APase.
• Isolation and characterization of Rhodococcus sp strains TNP2 and T12 that degrade 2,6,10,14-tetramethylpentadecane (pristane) at moderately low temperatures

  N Kunihiro, M Haruki, K Takano, M Morikawa, S Kanaya

  JOURNAL OF BIOTECHNOLOGY 115 (2) 129 - 136 0168-1656 2005/01 [Refereed][Not invited]
   

  Branched alkanes including 2,6,10,14-tetramethylpentadecane (pristane) are more resistant to biological degradation than straight-chain alkanes especially under low-temperature conditions, such as 10 degreesC. Two bacterial strains, TMP2 and T12, that are capable of degrading pristane at 10 degreesC were isolated and characterized. Both strains grew optimally at 30 degreesC and were identified as Rhodococcus sp. based on the 16S rRNA gene sequences. Strain T12 degraded comparable amounts of pristane in a range of temperatures from 10 to 30 degreesC and strain TMP2 degraded pristane similarly at 10 and 20 degreesC but did not degrade it at 30 degreesC. These data suggest that the strains have adapted their pristane degradation system to moderately low-temperature conditions. (C) 2004 Elsevier B.V. All rights reserved.
• Comparison of dibenzothiophene-degrading bacteria under low oxygen conditions

  Kitauchi F, Hirano S, Haruki M, Imanaka T, Morikawa M, Kanaya S

  J. Environ. Biotechnol. 環境バイオテクノロジー学会 4 (2) 117 - 120 1347-1856 2005 [Refereed][Not invited]
  
• Structure of RadB recombinase from a hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1: an implication for the formation of a near-7-fold helical assembly.

  Toshihiko Akiba, Noriyuki Ishii, Naeem Rashid, Masaaki Morikawa, Tadayuki Imanaka, Kazuaki Harata

  Nucleic acids research 33 (10) 3412 - 23 0305-1048 2005 [Refereed][Not invited]
   

  The X-ray crystal structure of RadB from Thermococcus kodakaraensis KOD1, an archaeal homologue of the RecA/Rad51 family proteins, have been determined in two crystal forms. The structure represents the core ATPase domain of the RecA/Rad51 proteins. Two independent molecules in the type 1 crystal were roughly related by 7-fold screw symmetry whereas non-crystallographic 2-fold symmetry was observed in the type 2 crystal. The dimer structure in the type 1 crystal is extended to construct a helical assembly, which resembles the filamentous structures reported for other RecA/Rad51 proteins. The molecular interface in the type 1 dimer is formed by facing a basic surface patch of one monomer to an acidic one of the other. The empty ATP binding pocket is located at the interface and barely concealed from the outside similarly to that in the active form of the RecA filament. The model assembly has a positively charged belt on one surface bordering the helical groove suitable for facile binding of DNA. Electron microscopy has revealed that, in the absence of ATP and DNA, RadB forms a filament with a similar diameter to that of the hypothetical assembly, although its helical properties were not confirmed.
• Kinetically robust monomeric protein from a hyperthermophile

  A Mukaiyama, K Takano, M Haruki, M Morikawa, S Kanaya

  BIOCHEMISTRY 43 (43) 13859 - 13866 0006-2960 2004/11 [Refereed][Not invited]
   

  Equilibrium and kinetic studies were carried out under denaturation conditions to clarify the energetic features of the high stability of a monomeric protein, ribonuclease HIT, from a hyperthermophile, Thermococcus kodakaraensis (Tk-RNase HII). Guanidine hydrochloride (GdnHCl)-induced unfolding and refolding were measured with circular dichroism at 220 nm, and heat-induced denaturation was studied with differential scanning calorimetry. Both GdnHCl- and heat-induced denaturation are very reversible. It was difficult to obtain the equilibrated unfolding curve of Tk-RNase HIT below 40 degreesC, because of the remarkably slow unfolding. The two-state unfolding and refolding reactions attained equilibrium at 50 degreesC after 2 weeks. The Gibbs energy change of GdnHCl-induced unfolding (DeltaG(H2O)) at 50 degreesC was 43.6 kJ mol(-1). The denaturation temperature in the DSC measurement shifted as a function of the scan rate; the denaturation temperature at a scan rate of 90 degreesC h(-1) was higher than at a scan rate of 5 degreesC h(-1). The unfolding and refolding kinetics of Tk-RNase HIT were approximated as a first-order reaction. The ln k(u) and ln k(r) values depended linearly on the denaturant concentration between 10 and 50 degreesC. The DeltaG(H2O) value obtained from the rate constant in water using the two-state model at 50 degreesC, 44.5 kJ mol(-1), was coincident with that from the equilibrium study, 43.6 kJ mol(-1), suggesting the two-state folding of Tk-RNase HII. The values for the rate constant in water of the unfolding for Tk-RNase HII were much smaller than those of E. coli RNase HI and Thermus thermophilus RNase HI, which has a denaturation temperature similar to that of Tk-RNase HII. In contrast, little difference was observed in the refolding rates among these proteins. These results indicate that the stabilization mechanism of monomeric protein from a hyperthermophile, Tk-RNase HII, with reversible two-state folding is characterized by remarkably slow unfolding.
• Cleavage of various peptides with pitrilysin from Escherichia coli: Kinetic analyses using beta-endorphin and its derivatives

  J Cornista, S Ikeuchi, M Haruki, A Kohara, K Takano, M Morikawa, S Kanaya

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 68 (10) 2128 - 2137 0916-8451 2004/10 [Refereed][Not invited]
   

  Pitrilysin from Escherichia coli was overproduced, purified, and analyzed for enzymatic activity using 14 peptides as a substrate. Pitrilysin cleaved all the peptides, except for two of the smallest, at a limited number of sites, but showed little amino acid specificity. It cleaved beta-endorphin (beta-EP) most effectively, with a K-m value of 0.36 mum and a k(cat) value of 750 min(-1). beta-EP consists of 31 residues and was predominantly cleaved by the enzyme at Lys(19)-Asn(20). Kinetic analyses using a series of beta-EP derivatives with N and/or C-terminal truncations and with amino acid substitutions revealed that three hydrophobic residues (Leu(14), Val(15), and Leu(17)) and the region 22-26 in beta-EP are responsible for high-affinity recognition by the enzyme. These two regions are located on the N- and C-terminal sides of the cleavage site in beta-EP, suggesting that the substrate binding pocket of pitrilysin spans its catalytic site.
• Gene cloning and biochemical characterizations of thermostable ribonuclease HIII from Bacillus stearothermophilus

  H Chon, R Nakano, N Ohtani, M Haruki, K Takano, M Morikawa, S Kanaya

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 68 (10) 2138 - 2147 0916-8451 2004/10 [Refereed][Not invited]
   

  The gene encoding RNase HIII from the thermophilic bacterium Bacillus stearothermophilus was cloned and overexpressed in Escherichia coli, and the recombinant protein (Bst-RNase HIII) was purified and biochemically characterized. Bst-RNase HIII is a monomeric protein with 310 amino acid residues, and shows an amino acid sequence identity of 47.1% with B. subtilis RNase HIII (Bsu-RNase HIII). The enzymatic properties of Bst-RNase HIII, such as pH optimum, metal ion requirement, and cleavage mode of the substrates, were similar to those of Bsu-RNase HIII However, Bst-RNase HIII was more stable than Bsu-RNase HIII, and the temperature (T-1/2) at which the enzyme loses half of its activity upon incubation for 10min was 55degreesC for Bst-RNase HIII and 35degreesC for Bsu-RNase HIII. The optimum temperature for Bst-RNase HIII activity was also shifted upward by roughly 20degreesC as compared to that of Bsu-RNase HIII. The availability of such a thermostable enzyme will facilitate structural studies of RNase HIII.
• Mutational and structural-based analyses of the osmolyte effect on protein stability

  K Takano, M Saito, M Morikawa, S Kanaya

  JOURNAL OF BIOCHEMISTRY 135 (6) 701 - 708 0021-924X 2004/06 [Refereed][Not invited]
   

  It is known that several naturally occurring substances known as osmolytes increase the conformational stability of proteins. Bolen and co-worker proposed the osmophobic theory, which asserts the osmolyte effect occurs because of an unfavorable interaction of osmolytes mainly with the protein backbone, based on the results on the transfer Gibbs energy of amino acids (Deltag) [Bolen and Baskakov (2001) J. Mol. Biol. 310, 955-963]. In this paper, we report the effect of sarcosine on the conformational stability (DeltaG) of RNase Sa (96 residues and one disulfide bond) and four mutant proteins. The thermal denaturation curves for RNase Sa in sarcosine fitted a two-state model on nonlinear least-squares analysis. All the RNase Sa proteins were stabilized by sarcosine. For example, the increase in stability of the wild-type protein in 4 M sarcosine due to the osmolyte effect (Delta(o)DeltaG) is 3.2 kcal/mol. Mutational analysis of the osmolyte effect indicated that the changed Delta(o)DeltaG values upon mutation (Delta(m)Delta(o)DeltaG), as estimated from the Deltag values, are similar to the experimental values. Structural-based analysis of the osmolyte effect was also performed using model denatured structures: (a) a fully extended model (single chain) with no disulfide bond, (b) two-part, unfolded models (two chains) with a disulfide bond constructed through molecular dynamic (MD) simulation, and (c) a two-part, folded model (two chains). The two-part, unfolded models were expected to be more suitable as denatured structures. The Delta(o)DeltaG values calculated using the two-part, unfolded models were more consistent with experimental values than those calculated using the fully extended and two-part, folded models. This suggests that MD simulation is useful for testing denatured structures. These results indicate that the osmophobic theory can explain the osmolyte effect on protein stability.
• Possible involvement of an FKBP family member protein from a psychrotrophic bacterium Shewanella sp SIB1 in cold-adaptation

  Y Suzuki, M Haruki, K Takano, M Morikawa, S Kanaya

  EUROPEAN JOURNAL OF BIOCHEMISTRY 271 (7) 1372 - 1381 0014-2956 2004/04 [Refereed][Not invited]
   

  A psychrotrophic bacterium Shewanella sp. strain SIB1 was grown at 4 and 20 degreesC, and total soluble proteins extracted from the cells were analyzed by two-dimensional polyacrylamide gel electrophoresis. Comparison of these patterns showed that the cellular content of a protein with a molecular mass of 28 kDa and an isoelectric point of four greatly increased at 4 degreesC compared to that at 20 degreesC. Determination of the N-terminal amino acid sequence, followed by the cloning and sequencing of the gene encoding this protein, revealed that this protein is a member of the FKBP family of proteins with an amino acid sequence identity of 56% to Escherichia coli FKBP22. This protein was overproduced in E. coli in a His-tagged form, purified, and analyzed for peptidyl-prolyl cis-trans isomerase activity. When this activity was determined by the protease coupling assay using N-succinyl-Ala-Leu-Pro-Phe-p-nitroanilide as a substrate at various temperatures, the protein exhibited the highest activity at 10 degreesC with a k(cat)/K-m value of 0.87 muM(-1).s(-1). When the peptidyl-prolyl cis-trans isomerase activity was determined by the RNase T-1 refolding assay at 10 and 20 degreesC, the protein exhibited higher activity at 10 degreesC with a k(cat)/K-m value of 0.50 muM(-1).s(-1). These k(cat)/K-m values are lower but comparable to those of E. coli FKBP22. We propose that a FKBP family protein is involved in cold-adaptation of psychrotrophic bacteria.
• Isolation and characterization of Xanthobacter polyaromaticivorans sp nov 127W that degrades polycyclic and heterocyclic aromatic compounds under extremely low oxygen conditions

  SI Hirano, F Kitauchi, M Haruki, T Imanaka, M Morikawa, S Kanaya

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 68 (3) 557 - 564 0916-8451 2004/03 [Refereed][Not invited]
   

  Two bacterial strains, 127W and T102, were isolated from anoxic crude oil tank sludge as effective degraders of dibenzothiophene (DBT), a model sulfur containing heterocyclic aromatic compound in crude oil. Strain 127W was more tolerant to oxygen limitation than T102 and was capable of degrading two- and three-ring polycyclic and heterocyclic aromatic compounds under both aerobic and low oxygen conditions. Strain 127W degraded 0.082, 0.055, and 0.064 mm of DBT, naphthalene, and anthracene, respectively, in one week with dissolved oxygen </=0.2ppm (0.006mm). Degradation by 127W cell-free extracts for DBT was increased by addition of sodium hydrogencarbonate under this oxygen concentration. Phylogenetic analysis of the 16S rRNA gene sequence and physiological characteristics indicate that the strains 127W and T102 belong to new species of the genus Xanthobacter and Pseudomonas stutzeri, respectively. We propose X. polyaromaticivorans sp. nov. 127W.
• 次世代のペプチド合成法

  森川正章, 金谷茂則

  日本化学会バイオテクノロジー部会ニュースレター 8 5 - 7 2004 [Refereed][Not invited]
  
• Description of Thermococcus kodakaraensis sp. nov., a well studied hyperthermophilic archaeon previously reported as Pyrococcus sp. KOD1

  Haruyuki Atomi, Toshiaki Fukui, Tamotsu Kanai, Masaaki Morikawa, Tadayuki Imanaka

  Archaea 1 (4) 263 - 267 1472-3646 2004 [Refereed][Not invited]
   

  A hyperthermophilic archaeal strain, KOD1, isolated from a solfatara on Kodakara Island, Japan, has previously been reported as Pyrococcus sp. KOD1. However, a detailed phylogenetic tree, made possible by the recent accumulation of 16S rRNA sequences of various species in the order Thermococcales, indicated that strain KOD1 is a member of the genus Thermococcus. We performed DNA-DNA hybridization tests against species that displayed high similarity in terms of 16S ribosomal DNA sequences, including Thermococcus peptonophilus and Thermococcus stetteri. Hybridization results and differences in growth characteristics and substrate utilization differentiated strain KOD1 from T. peptonophilus and T. stetteri at the species level. Our results indicate that strain KOD1 represents a new species of Thermococcus, which we designate as Thermococcus kodakaraensis KOD1 sp. nov.
• Cloning and characterization of the gene cluster encoding arthrofactin synthetase from Pseudomonas sp MIS38

  N Roongsawang, K Hase, M Haruki, T Imanaka, M Morikawa, S Kanaya

  CHEMISTRY & BIOLOGY 10 (9) 869 - 880 1074-5521 2003/09 [Refereed][Not invited]
   

  Arthrofactin is a potent cyclic lipopeptide-type biosurfactant produced by Pseudomonas sp. MIS38. In this work, an arthrofactin synthetase gene cluster (arf) spanning 38.7 kb was cloned and characterized. Three genes termed arfA, arfB, and arfC encode ArfA, ArfB, and ArfC, containing two, four, and five functional modules, respectively. Each module bears condensation, adenylation, and thiolation domains, like other nonribosomal peptide synthetases. However, unlike most of them, none of the 11 modules possess the epimerization domain responsible for the conversion of amino acid residues from L to D form. Possible L-and D-Leu adenylation domains specifically recognized only L-Leu. Moreover, two thioesterase domains are tandemly located at the C-terminal end of ArfC. These results suggest that ArfA, ArfB, and ArfC assemble to form a unique structure. Gene disruption of arfB impaired arthrofactin production, reduced swarming activity, and enhanced biofilm formation.
• Production and characterization of biosurfactants from Bacillus licheniformis F2.2

  J Thaniyavarn, N Roongsawang, T Kameyama, M Haruki, T Imanaka, M Morikawa, S Kanaya

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 67 (6) 1239 - 1244 0916-8451 2003/06 [Refereed][Not invited]
   

  A biosurfactant-producing strain, Bacillus licheniformis F2.2, was isolated from a fermented food in Thailand. The strain was capable of producing a new biosurfactant, BL1193, as well as two kinds of popular lipopeptide biosurfactants, plipastatin and surfactin. Mass spectrometry and FT-IR analysis indicated that BL1193 had a molecular mass of 1,193 Da with no peptide portion in the molecule. While plipastatin and surfactin were abundantly produced in a nutrient YPD medium, BL1193 was produced only in a synthetic DF medium containing no amino acids. According to an oil displacement activity test, the specific activity of BL1193 (6.53 kBS units/mg) is equivalent to that of surfactin (5.78-6.83 kBS units/mg).
• Dispensability of glutamic acid 48 and aspartic acid 134 for Mn2+-dependent activity of Escherichia coli ribonuclease HI

  Y Tsunaka, M Haruki, M Morikawa, M Oobatake, S Kanaya

  BIOCHEMISTRY 42 (11) 3366 - 3374 0006-2960 2003/03 [Refereed][Not invited]
   

  The activities of the eight mutant proteins of Escherichia coli RNase HI, in which the four carboxylic amino acids (Asp(10), Glu(48), Asp(70), and Asp(134)) involved in catalysis are changed to Asn (Gln) or Ala, were examined in the presence of Mn2+. Of these proteins, the E48A, E48Q, D134A, and D134N proteins exhibited the activity, indicating that Glu(48) and Asp(134) are dispensable for Mn2+-dependent activity. The maximal activities of the E48A and D134A proteins were comparable to that of the wild-type protein. However, unlike the wild-type protein, these mutant proteins exhibited the maximal activities in the presence of > 100 muM MnCl2, and their activities were not inhibited at higher Mn2+ concentrations (up to 10 mM). The wild-type protein contains two Mn2+ binding sites and is activated upon binding of one Mn2+ ion at site 1 at low (similar to1 muM) Mn2+ concentrations. This activity is attenuated upon binding of a second Mn2+ ion at site 2 at high (> 10 muM) Mn2+ concentrations. The cleavage specificities of the mutant proteins, which were examined using oligomeric substrates at high Mn2+ concentrations, were identical to that of the wild-type protein at low Mn2+ concentrations but were different from that of the wild-type protein at high Mn2+ concentrations. These results suggest that one Mn2+ ion binds to the E48A, E48Q, D134A, and D134N proteins at site 1 or a nearby site with weaker affinities. The binding analyses of the Mn2+ ion to these proteins in the absence of the substrate support this hypothesis. When Mn2+ ion is used as a metal cofactor, the Mn2+ ion itself, instead of Glu(48) and Asp(134), probably holds water molecules required for activity.
• Crystallization and preliminary X-ray analysis of TBP-interacting protein from the hyperthermophilic archaeon Thermococcus kodakaraensis strain KOD1

  T Yamamoto, T Matsuda, N Sakamoto, H Matsumura, T Inoue, M Morikawa, S Kanaya, Y Kai

  ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY 59 (2) 372 - 374 0907-4449 2003/02 [Refereed][Not invited]
   

  The 26 kDa TBP (TATA-binding protein) interacting protein from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 (Tk-TIP26) is a possible transcriptional regulatory protein in Thermococcales. Here, the crystallization of both the native and selenomethionine-substituted proteins and data collection are described. The native crystals belong to the tetragonal space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = 73.83, c = 86.41 Angstrom, and diffract to 2.2 Angstrom using synchrotron radiation. MAD data was collected and a Bijvoet difference Patterson map showed strong peaks sufficient to determine the positions of the Se atoms.
• 掘り起こせ宝の山−微生物のしたたかな戦略バイオフィルム

  森川 正章

  バイオサイエンスとインダストリー 61 2003 [Refereed][Not invited]
  
• Biofilms-Even Microorganisms Live Together

  MORIKAWA Masaaki

  KAGAKU TO SEIBUTSU Japan Society for Bioscience, Biotechnology, and Agrochemistry 41 (1) 32 - 37 0453-073X 2003 [Refereed][Not invited]
  
• Application of a two-liquid system to sitting-drop vapour-diffusion protein crystallization

  H Adachi, K Takano, M Morikawa, S Kanaya, M Yoshimura, Y Mori, T Sasaki

  ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY 59 (Pt1) 194 - 196 0907-4449 2003/01 [Refereed][Not invited]
   

  A new method of protein crystallization, the floating-drop vapour-diffusion technique, has been developed. The method combines the traditional sitting-drop vapour-diffusion technique and a novel two-liquid system. A crystallization drop composed of a mixture of sample and reagent floats on an insoluble and very dense liquid. Protein crystals are grown by vapour diffusion at the interface of the two liquids. The method makes it possible to remove the crystals easily without causing mechanical damage. This approach also significantly reduces the time and cost compared with the hanging-drop technique, which is presently the most popular method for protein crystallization.
• Laser irradiated growth of protein crystal.

  Adachi H, Takano K, Hosokawa Y, Inoue T, Mori Y, Matsumura H, Yoshimura M, Tsunaka Y, Morikawa M, Kanaya S, Masuhara H, Kai Y, Sasaki T

  Jpn. J. Appl. Phys. Japan Society of Applied Physics 42 (7B) L798 - L800 0021-4922 2003 [Refereed][Not invited]
   

  We succeeded in the first ever generation of protein crystals by laser irradiation. We call this process Laser Irradiated Growth Technique (LIGHT). Effective crystallization was confirmed by applying an intense femtosecond laser. The crystallization period was dramatically shortened by LIGHT. In addition, protein crystals were obtained by LIGHT from normally uncrystallized conditions. These results indicate that intense femtosecond laser irradiation generates crystal nuclei; protein crystals can then be grown from the nuclei that act as seeds in a supersaturated solution. The nuclei formation is possible primarily due to nonlinear nucleation processes of an intense femtosecond laser with a peak intensity of over a gigawatt (GW).
• Stability of Ribonuclease HII from Hyperthermophile, Thermococcus kod akaraensis

  Mukaiyama A., Takano K., Haruki M., Morikawa M., Kanaya S.

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 43 S68  2003
• Isolation and characterization of a halotolerant Bacillus subtilis BBK-1 which produces three kinds of lipopeptides: bacillomycin L, plipastatin, and surfactin

  N Roongsawang, J Thaniyavarn, S Thaniyavarn, T Kameyama, M Haruki, T Imanaka, M Morikawa, S Kanaya

  EXTREMOPHILES 6 (6) 499 - 506 1431-0651 2002/12 [Refereed][Not invited]
   

  Twenty-three halotolerant and biosurfactant producing strains were collected from salty conditions in central Thailand. One of the strains designated BBK-1 produced the biosurfactants with the highest activity. BBK-1 was isolated from fermented foods and was identified as B. subtilis based on its physiological characteristics and 16S rRNA gene sequence. We show that the strain grows in media containing NaCl up to 16% (w/v) and produces biosurfactants in NaCl up to 8%. We found that B. subtilis BBK-1 produces three kinds of surface-active lipopeptides simultaneously. By their respective molecular weights and amino acid compositions, it is indicated that these lipopeptides are bacillomycin L, plipastatin, and surfactin. In order to analyze the production mechanism of lipopeptides further in the strain, a generally important biosynthetic gene encoding 4'-phosphopantetheinyl transferase was cloned and sequenced. The gene existed in a single copy in the genome and the deduced amino acid sequence was almost identical to that of Lpa-14 from B. subtilis strain RB14, which co-produces iturin A and surfactin.
• Oleomonas sagaranensis gen. nov., sp nov., represents a novel genus in the alpha-Proteobacteria

  T Kanamori, N Rashid, M Morikawa, H Atomi, T Imanaka

  FEMS MICROBIOLOGY LETTERS 217 (2) 255 - 261 0378-1097 2002/12 [Refereed][Not invited]
   

  A Gram-negative bacterium was previously isolated from an oil field in Shizuoka, Japan, and designated strain HD-1. Here we have performed detailed characterization of the strain, and have found that it represents a novel genus. The 16S rRNA sequence of strain HD-1 displayed highest similarity to various uncultured species (86.7 similar to 99.7%), along with 86.2 similar to 88.2% similarity to sequences from Azospirillum, Methylobacterium, Rhizobium, and Hyphomicrobium, all members of the alpha-Proteobacteria. Phylogenetic analysis revealed that HD-1 represented a deep-branched lineage among the alpha-Proteobacteria. DNA-DNA hybridization analysis with Azospirillum lipoferum and Hyphomicrobium vulgare revealed low levels of similarity among the strains. We further examined the biochemical properties of the strain under aerobic conditions. Among carbon sources, ethanol, n-propanol, n-butanol, and n-tetradecanol were the most preferred, while acetate, propionate, and pyruvate also supported high levels of growth. The strain could also grow on aromatic compounds such as toluene, benzene and phenol, and aliphatic hydrocarbons such as n-octane and n-tetradecane. In contrast, glycerol and various sugars, including glucose, fructose, maltose, and lactose, failed to support growth of HD-1. Under an anaerobic gas phase with butanol as the carbon source, little increase in cell weight was observed with the addition of several possible electron acceptors. As strain HD-1 represents a novel genus in the a-Proteobacteria, we designated the strain as Oleomonas sagaranensis gen. nov., sp. nov., strain HD-1. (C) 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
• Cleavage of a DNA-RNA-DNA/DNA chimeric substrate containing a single ribonucleotide at the DNA-RNA junction with prokaryotic RNases HII

  M Haruki, Y Tsunaka, M Morikawa, S Kanaya

  FEBS LETTERS 531 (2) 204 - 208 0014-5793 2002/11 [Refereed][Not invited]
   

  We have analyzed the cleavage specificities of various prokaryotic Type 2 ribonucleases H (RNases H) on chimeric DNA-RNA-DNA/DNA substrates containing one to four ribonucleotides. RNases HII from Bacillus subtilis and Thermococcus kodakaraensis cleaved all of these substrates to produce a DNA segment with a 5'-monoribonucleotide. Consequently, these enzymes cleaved even the chimeric substrate containing a single ribonucleotide at the DNA-RNA junction (5'-side of the single ribonucleotide). In contrast, Escherichia coli RNase HI and B. subtilis RNase HIII did not cleave the chimeric substrate containing a single ribonucleotide. These results suggest that bacterial and archaeal RNases MI are involved in excision of a single ribonucleotide misincorporated into DNA. (C) 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.
• Site-specific cleavage of MS2 RNA by a thermostable DNA-linked RNase H

  H Chon, Y Tsunaka, M Haruki, M Morikawa, S Kanaya

  PROTEIN ENGINEERING 15 (8) 683 - 688 0269-2139 2002/08 [Refereed][Not invited]
   

  A series of DNA-linked RNases H, in which the 15-mer DNA is cross-linked to the Thermus thermophilus RNase HI (TRNH) variants at positions 135, 136, 137 and 138, were constructed and analyzed for their abilities to cleave the complementary 15-mer RNA. Of these, that with the DNA adduct at position 135 most efficiently cleaved the RNA substrate, indicating that position 135 is the most appropriate cross-linking site among those examined. To examine whether DNA-linked RNase H also site-specifically cleaves a highly structured natural RNA, DNA-linked TRNHs with a series of DNA adducts varying in size at position 135 were constructed and analyzed for their abilities to cleave MS2 RNA. These DNA adducts were designed such that DNA-linked enzymes cleave MS2 RNA at a loop around residue 2790. Of the four DNA-linked TRNHs with the 8-, 12-, 16- and 20-mer DNA adducts, only that with the 16-mer DNA adduct efficiently and site-specifically cleaved MS2 RNA. Primer extension revealed that this DNA-linked TRNH cleaved MS2 RNA within the target sequence.
• Role of repetitive nine-residue sequence motifs in secretion, enzymatic activity, and protein conformation of a family I.3 lipase

  HJ Kwon, M Haruki, M Morikawa, K Omori, S Kanaya

  JOURNAL OF BIOSCIENCE AND BIOENGINEERING 93 (2) 157 - 164 1389-1723 2002/02 [Refereed][Not invited]
   

  A family 1.3 lipase from Pseudomonas sp. MIS38 (PML) contains 12 repeats of a nine-residue sequence motif in the C-terminal region. To elucidate the role of these repetitive sequences, mutant proteins PML5, PML4, PML1, and PML0, in which 7, 8, 11, and all 12 of the repetitive sequences are deleted, and PMLDelta19, in which 19 C-terminal residues are truncated, were constructed. Escherichia coli DH5 cells carrying the Serratia marcescens Lip system permitted the secretion of the wild-type and all of the mutant proteins except for PMLDelta19, although they were partially accumulated in the cells in an insoluble form as well. Both the secretion level and cellular content of the proteins decreased in the order PML>PML5>PML4>PML1>PML0, indicating that repetitive sequences are not required for secretion of PML but are important for its stability in the cells. All the mutant proteins were purified in a refolded form and their biochemical properties were characterized. CD spectra, the Ca2+ contents, and susceptibility to chymotryptic digestion strongly suggested that the five repetitive sequences remaining in PML5 are sufficient to form a P-roll structure, whereas the four in PML4 are not. PML5 and PMLDelta19 showed both lipase and esterase activities, whereas PML4, PML1, and PML0 were inactive. These results suggest that the enzymatic activity of PML is not seriously affected by a deletion or truncation at the C-terminal region as long as a succession of repetitive sequences can build a beta-roll structure.
• Importance of an N-terminal extension in ribonuclease HII from Bacillus stearothermophilus for substrate binding

  A Muroya, R Nakano, N Ohtani, M Haruki, M Morikawa, S Kanaya

  JOURNAL OF BIOSCIENCE AND BIOENGINEERING 93 (2) 170 - 175 1389-1723 2002/02 [Refereed][Not invited]
   

  The gene encoding ribonuclease HII from Bacillus stearothermophilus was cloned and expressed in Escherichia coli. The overproduced protein, Bst-RNase HII, was purified and biochemically characterized. Bst-RNase HII, which consists of 259 amino acid residues, showed the highest amino acid sequence identity (50.2%) to Bacillus subtilis R-Nase HII. Like B. subtilis RNase HII, it exhibited Mn2+-dependent RNase H activity. It was, however, more thermostable than B. subtilis RNase HII. When the Bst-RNase HII amino acid sequence is compared with that of Thermococcus kodakaraensis RNase HII, to which it shows 29.8% identity, 30 residues are observed to be truncated from the C-terminus and there is an extension of 71 residues at the N-terminus. The C-terminal truncation results in the loss of the alpha9 helix, which is rich in basic amino acid residues and is therefore important for substrate binding. A truncated protein, Delta59-Bst-RNase HII, in which most of the N-terminal extension was removed, completely lost its RNase H activity. Surface plasmon resonance analysis indicated that this truncated protein did not bind to the substrate. These results suggest that the N-terminal extension of Bst-RNase HII is important for substrate binding. Because B. subtilis RNase HII has an N-terminal extension of the same length and these extensions contain a region in which basic amino acid residues are clustered, the Bacillus enzymes may represent a novel type of RNase H which possesses a substrate-binding domain at the N-terminus.
• 特殊環境微生物の環境修復への応用と限界

  森川 正章

  創造と実践—大阪大学全学共通教育機構— (2) 66 - 71 2002 [Refereed][Not invited]
  
• Heat labile ribonuclease HI from a psychrotrophic bacterium: gene cloning, characterization and site-directed mutagenesis

  N Ohtani, M Haruki, M Morikawa, S Kanaya

  PROTEIN ENGINEERING 14 (12) 975 - 982 0269-2139 2001/12 [Refereed][Not invited]
   

  The rnhA gene encoding RNase HI from a psychrotrophic bacterium, Shewanella sp. SIB1, was cloned, sequenced and overexpressed in an rnh mutant strain of Escherichia coli. SIB1 RNase HI is composed of 157 amino acid residues and shows 63% amino acid sequence identity to E.coli RNase HI. Upon induction, the recombinant protein accumulated in the cells in an insoluble form. This protein was solubilized and purified in the presence of 7 M urea and refolded by removing urea. Determination of the enzymatic activity using M13 DNA-RNA hybrid as a substrate revealed that the enzymatic properties of SIB 1 RNase HI, such as divalent cation requirement, pH optimum and cleavage mode of a substrate, are similar to those of E.coli RNase HI. However, SIB1 RNase HI was much less stable than E.coli RNase HI and the temperature (T-1/2) at which the enzyme loses half of its activity upon incubation for 10 min was similar to25degreesC for SIB1 RNase HI and similar to60degreesC for E.coli RNase HI. The optimum temperature for the SIB1. RNase HI activity was also shifted downward by 20 C compared with that of E.coli RNase HI. Nevertheless, SIB1. RNase HI was less active than E.coli RNase HI even at low temperatures. The specific activity determined at 10 C was 0.29 units/mg for SIB1 RNase HI and 1.3 units/mg for E.coli RNase HI. Site-directed mutagenesis studies suggest that the amino acid substitution in the middle of the alphaI-helix (Pro52 for SIB1 RNase HI and Ala52 for E.coli RNase HI) partly accounts for the difference in the stability and activity between SIB1 and E.coli RNases HI.
• Ca2+-induced folding of a family 1.3 lipase with repetitive Ca2+ binding motifs at the C-terminus

  K Amada, HJ Kwon, M Haruki, M Morikawa, S Kanaya

  FEBS LETTERS 509 (1) 17 - 21 0014-5793 2001/11 [Refereed][Not invited]
   

  In order to understand a role of the Ca2+ ion on the structure and function of a Ca2+-dependent family 1.3 lipase from Pseudomonas sp. MIS38, apo-PML, holo-PML, holo-PML*, and the N-terminal domain alone (N-fragment) were prepared and biochemically characterized. Apo-PML and holo-PML represent refolded proteins in the absence and presence of the Ca2+ ion, respectively. Holo-PML* represents a holo-PML dialyzed against 20 mM Tris-HCl (pH 7.5). The results suggest that the C-terminal domain of PML is almost fully unfolded in the apo-form and its folding is induced by Ca2+ binding. The folding of this C-terminal domain may be required to make a conformation of the N-terminal catalytic domain functional. (C) 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.
• Isolation and characterization of psychrotrophic bacteria from oil-reservoir water and oil sands

  T Kato, M Haruki, T Imanaka, M Morikawa, S Kanaya

  APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 55 (6) 794 - 800 0175-7598 2001/06 [Refereed][Not invited]
   

  Four psychrotrophic strains, which grew at 4 degreesC but not at 37 degreesC, were isolated from Japanese oil-reservoir water (strains SIB1, SIC1, SIS1) and Canadian oil sands (strain CAB1). Strains SIB1, SIS1, and CAB1 had a maximum growth rate at 20 degreesC and grew to the highest cell densities at the cultivation temperature of 0-4 degreesC. Strain SIS1 was capable of growing even at -5 degreesC. The growth profile of strain SIC I was rather similar to that of a mesophilic bacterium. Strains SIB1, SIC1, and SIS1 were identified as members of the genus Shewanella, and strain CAB1 was a member of the genus Arthrobacter. All these strains exhibited weak degradation ability against catechol, a hydroxylated aromatic hydrocarbon, and tributyrin. These strains are expected to be of potential use in the in situ bioremediation technology of hazardous hydrocarbons and esters under low-temperature conditions.
• Active subtilisin-like protease from a hyperthermophilic archaeon in a form with a putative prosequence

  Y Kannan, Y Koga, Y Inoue, M Haruki, M Takagi, T Imanaka, M Morikawa, S Kanaya

  APPLIED AND ENVIRONMENTAL MICROBIOLOGY 67 (6) 2445 - 2452 0099-2240 2001/06 [Refereed][Not invited]
   

  The gene encoding subtilisin-like protease T, kodakaraensis subtilisin was cloned from a hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. T. kodakaraensis subtilisin is a member of the subtilisin family and composed of 422 amino acid residues with a molecular weight of 43,783, It consists of a putative presequence, prosequence, and catalytic domain. Like bacterial subtilisins, T. kodakaraensis subtilisin was overproduced in Escherichia coli in a form with a putative prosequence in inclusion bodies, solubilized in the presence of 8 M urea, and refolded and converted to an active molecule. However, unlike bacterial subtilisins, in which the prosequence was removed from the catalytic domain by autoprocessing upon refolding, T, kodakaraensis subtilisin was refolded in a form with a putative prosequence. This refolded protein of recombinant T. kodakaraensis subtilisin which is composed of 398 amino acid residues (Gly(-82) to Gly(316)), was purified to give a single band on a sodium dodecyl sulfate (SDS)-polyacrylamide gel and characterized for biochemical and enzymatic properties. The good agreement of the molecular weights estimated by SDS-polyacrylamide gel electrophoresis (44,000) and gel filtration (40,000) suggests that T. kodakaraensis subtilisin exists in a monomeric form. T. kodakaraensis subtilisin hydrolyzed the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide only in the presence of the Ca2+ ion with an optimal pH and temperature of pH 9.5 and 80 degreesC, Like bacterial subtilisins, it showed a broad substrate specificity, with a preference for aromatic or large nonpolar P1 substrate residues. However, it was much more stable than bacterial subtilisins against heat inactivation and lost activity with half-lives of > 60 min at 80 degreesC, 20 min at 90 degreesC, and 7 min at 100 degreesC.
• Interaction of TIP26 from a hyperthermophilic archaeon with TFB/TBP/DNA ternary complex

  T Matsuda, R Fujikawa, M Haruki, XF Tang, S Ezaki, T Imanaka, M Morikawa, S Kanaya

  EXTREMOPHILES 5 (3) 177 - 182 1431-0651 2001/06 [Refereed][Not invited]
   

  Interactions of TBP-interacting protein (TIP26), TBP, and TFB from a hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 with TATA-DNA were examined by electrophoretic mobility shift assay. Tk-TFB formed a ternary complex with Tk-TBP and TATA-DNA. Tk-TIP26 did not inhibit the formation of this ternary complex, but interacted with it to form a TIP26/TFB/TBP/DNA quaternary complex. This interaction is rather weak, and a large excess of Tk-TIP26 over Tk-TBP is required to fully convert the TFB/TBP/DNA ternary complex to the quaternary complex. However, determination of the concentration of Tk-TIP26 and Tk-TBP in KOD1 cells by Western blotting analysis indicated that the concentration of Tk-TIP26 is approximately ten times that of Tk-TBP, suggesting that the quaternary complex might also form in vivo.
• Stabilities of chimeras of hyperthermophilic and mesophilic glycerol kinases constructed by DNA shuffling

  Y Koga, M Haruki, M Morikawa, S Kanaya

  JOURNAL OF BIOSCIENCE AND BIOENGINEERING 91 (6) 551 - 556 1389-1723 2001/06 [Refereed][Not invited]
   

  Glycerol kinases from Thermococcus kodakaraensis KOD1 (Tk-GK) and Escherichia coli (Ec-GK) greatly differ in thermostability. The temperature (T-1/2) at which the enzymes lose half of their activity upon incubation for 20 min is 50-55 degreesC for Ec-GK and similar to 95 degreesC for Tk-GK. To examine whether the amino acid substitutions that make Tk-GK more stable than Ec-GK are localized in a limited region, the chimeras of two parental genes encoding Tk-GK and Ec-GK were constructed by DNA shuffling. E. coli cells were transformed with a plasmid library harboring these chimeras and screened for those that produce chimeric enzymes which are more stable than Ec-GK. Four chimeric enzymes were isolated and purified, and their biochemical properties characterized. Replacement of 83 or 93 residues in the C-terminus of Ec-GK with the corresponding ones of Tk-GK increased the T-1/2 value of Ec-GK by 25-30 degreesC. In contrast, replacement of 85 residues in the N-terminus of Ec-GK with the corresponding ones of Tk-GK reduced the T-1/2 value by 5-10 degreesC. In addition, replacement of 10 residues in the C-terminus of Tk-GK with the corresponding ones of Ec-GK reduced the T-1/2 value of Tk-GK by similar to 15 degreesC. Measurement of the far-UV CD spectra indicates that the three-dimensional structures of the chimeric enzymes, as well as those of the parent enzymes, are similar to one another. These results suggest that the amino acid substitutions responsible for the high stability of Tk-GK are largely localized in the C-terminal region.
• Erratum - Identification of the histidine and aspartic acid residues essential for enzymatic activity of a family I.3 lipase by site-directed mutagenesis (FEBS Letters (2000) 483 (139-142) PII: S0014579300021037)

  H. J. Kwon, K. Amada, M. Haruki, M. Morikawa, S. Kanaya

  FEBS Letters 497 (2-3) 174  0014-5793 2001/05/25 [Refereed][Not invited]
  
• Strong nucleic acid binding to the Escherichia coli RNase HI mutant with two arginine residues at the active site

  Y Tsunaka, M Haruki, M Morikawa, S Kanaya

  BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY 1547 (1) 135 - 142 0167-4838 2001/05 [Refereed][Not invited]
   

  The biochemical properties of the mutant protein D10R/E48R of Escherichia coli RNase HI, in which Asp(10) and Glu(48) are both replaced by Arg, were characterized. This mutant protein has been reported to have metal-independent RNase H activity at acidic pH [Casareno et al. (1995) J. Am. Chem. Sec. 117, 11011-11012]. The far- and near-UV CD spectra of this mutant protein were similar to those of the wild-type protein, suggesting that the protein conformation is not markedly changed by these mutations. Nevertheless, we found that this mutant protein did not show any RNase H activity in vitro. Instead, it showed high-nucleic-acid-binding affinity. Protein footprinting analyses suggest that DNA/RNA hybrid binds to or around the presumed substrate-binding site of the protein. In addition, this mutant protein did not complement the temperature-sensitive growth phenotype of the rnhA mutant strain, E. coli MIC3001, even at pH 6.0, suggesting that it does not show RNase H activity in vivo as well. These results are consistent with a current model for the catalytic mechanism of the enzyme, in which Glu(48) is not responsible for Mg2+ binding but is involved in the catalytic function. (C) 2001 Elsevier Science B.V. All rights reserved.
• Catalytic center of an archaeal type 2 ribonuclease H as revealed by X-ray crystallographic and mutational analyses

  A Muroya, D Tsuchiya, M Ishikawa, M Haruki, M Morikawa, S Kanaya, K Morikawa

  PROTEIN SCIENCE 10 (4) 707 - 714 0961-8368 2001/04 [Refereed][Not invited]
   

  The catalytic center of an archaeal Type 2 RNase H has been identified by a combination of X-ray crystallographic and mutational analyses. The crystal structure of the Type 2 RNase H from Thermococcus kodakaraensis KOD1 has revealed that the N-terminal major domain adopts the RNase H fold, despite the poor sequence similarity to the Type 1 RNase H. Mutational analyses showed that the catalytic reaction requires four acidic residues, which are well conserved in the Type 1 RNase H and the members of the polynucleotidyl transferase family. Thus, the Type 1 and Type 2 RNases H seem to share a common catalytic mechanism, except for the requirement of histidine as a general base in the former enzyme. Combined with the results from deletion mutant analyses, the structure suggests that the C-terminal domain of the Type 2 RNase H is involved in the interaction with the DNA/RNA hybrid.
• バイオレメディエーション

  森川 正章

  学研ハイベスト教科事典「生物と環境」 223  2001 [Refereed][Not invited]
  
• Thiol protease from Thermococcus kodakaraensis KOD1

  M Morikawa, T Imanaka

  HYPERTHERMOPHILIC ENZYMES, PT A 330 424 - 433 0076-6879 2001 [Refereed][Not invited]
  
• Isolation and characterization of long-chain-alkane degrading Bacillus thermoleovorans from deep subterranean petroleum reservoirs

  T Kato, M Haruki, T Imanaka, M Morikawa, S Kanaya

  JOURNAL OF BIOSCIENCE AND BIOENGINEERING 91 (1) 64 - 70 1389-1723 2001/01 [Refereed][Not invited]
   

  Two extremely thermophilic alkane-degrading bacterial strains, B23 and H41, were respectively isolated from deep subterranean petroleum reservoirs in the Minami-aga (Niigata) and Yabase (Akita) oil fields. Both strains were able to grow at temperatures ranging from 50 to 80 degreesC, with optimal growth at 70 degreesC for B23 and 65 degreesC for H41. From 16S rRNA gene sequence analysis and physiological characterization, both strains were identified as Bacillus thermoleovorans (identities of 99.5% and 99.6% to strain DSM 5366, and 98.3% and 98.7% to the type strain LEH-1(TS), respectively). Strains B23 and H41 effectively (>60%) degraded n-alkanes longer than C12 and C15, respectively, at 70 degreesC, while strain LEH-1(TS) degraded undecane (C11) most effectively. When B23 and H41 were cultivated in the presence of heptadecane, heptadecanoate and pentadecanoate were specifically accumulated in the cells. These results strongly suggest that the two strains degraded n-alkanes by a terminal oxidation pathway, followed by a beta -oxidation pathway.
• Gene cloning of an alcohol dehydrogenase from thermophilic alkane-degrading Bacillus thermoleovorans B23

  T Kato, A Miyanaga, M Haruki, T Imanaka, M Morikawa, S Kanaya

  JOURNAL OF BIOSCIENCE AND BIOENGINEERING 91 (1) 100 - 102 1389-1723 2001/01 [Refereed][Not invited]
   

  The gene encoding an alcohol dehydrogenase (Bt-ADH) was cloned from a newly isolated thermophilic alkane-degrading Bacillus thermoleovorans, strain B23. The gene conferred 1-tetradecanol dehydrogenase activity on Escherichia coli cells. Bt-ADH is composed of 249 amino acid residues and the calculated molecular mass is 27,196 Da. A tyrosine residue in the active site and a glycine-rich sequence (GGXXGI/LG) constituting probable nicotinamide adenine dinucleotide (NAD(+)) or nicotinamide adenine dinucleotide phosphate (NADP(+)) binding site were completely conserved in the Bt-ADH sequence at positions 155 and 11, respectively. A phylogenetic analysis of Bt-ADH suggested that the enzyme belongs to the zinc-independent ADH Group II. Its highest similarity (48% identical) was to a hypothetical oxidoreductase from a hyperthermophile, Thermotoga maritima.
• RecA/Rad51 homolog from Thermococcus kodakaraensis KOD1

  N Rashid, M Morikawa, S Kanaya, H Atomi, T Imanaka

  HYPERTHERMOPHILIC ENZYMES, PT C 334 261 - 270 0076-6879 2001 [Refereed][Not invited]
  
• Efficient cleavage of RNA at high temperatures by a thermostable DNA-linked ribonuclease H

  M Haruki, T Nogawa, N Hirano, H Chon, Y Tsunaka, M Morikawa, S Kanaya

  PROTEIN ENGINEERING 13 (12) 881 - 886 0269-2139 2000/12 [Refereed][Not invited]
   

  To construct a DNA-linked RNase H, which cleaves RNA site-specifically at high temperatures, the 15-mer DNA, which is complementary to the polypurine-tract sequence of human immunodeficiency virus-1 RNA (PPT-RNA), was cross-linked to the unique thiol group of Cys135 in the Thermus thermophilus RNase HI variant. The resultant DNA-linked enzyme (d15-C135/TRNH), as well as the d15-C135/ERNH, in which the RNase H portion of the d15C135/TRNH is replaced by the Escherichia coli RNase HI variant, cleaved the 15-mer PPT-RNA site-specifically. The mixture of the unmodified enzyme and the unlinked 15-mer DNA also cleaved the PPT-RNA but in a less strict manner, In addition, this mixture cleaved the PIT-RNA much less effectively than the DNA-linked enzyme. These results indicate that the cross-linking limits but accelerates the interaction between the enzyme and the DNA/RNA substrate. The d15-C135/TRNH cleaved the PPT-RNA more effectively than the d15-C135/ERNH at temperatures higher than 50 degreesC, The d15-C135/TRNH showed the highest activity at 65 degreesC, at which the d15-C135/ERNH showed little activity. Such a thermostable DNA-linked RNase H may be useful to cleave RNA molecules with highly ordered structures in a sequence-specific manner.
• Catalysis by Escherichia coli ribonuclease HI is facilitated by a phosphate group of the substrate

  M Haruki, Y Tsunaka, M Morikawa, S Iwai, S Kanaya

  BIOCHEMISTRY 39 (45) 13939 - 13944 0006-2960 2000/11 [Refereed][Not invited]
   

  To investigate the role of the phosphate group 3' to the scissile phosphodiester bond of the substrate in the catalytic mechanism of Escherichia coli ribonuclease HI (RNase HI), we have used modified RNA-DNA hybrid substrates carrying a phosphorothioate substitution at this position or lacking this phosphate group for the cleavage reaction. Kinetic parameters of the H(124)A mutant enzyme, in which His(124) was substituted with Ala, as well as those of the wild-type RNase HI, were determined. Substitution of the pro-R-p-oxygen of the phosphate group 3' to the scissile phosphodiester bond of the substrate with sulfur reduced the k(cat) value of the wild-type RNase HI by 6.9-fold and that of the H(124)A mutant enzyme by only 1.9-fold. In contrast, substitution of the pro-Sb-oxygen of the phosphate group at this position with sulfur had little effect on the k(cat) value of the wild-type and H(124)A mutant enzymes. The results obtained for the substrate lacking this phosphate group were consistent with those obtained for the substrates with the phosphorothioate substitutions. In addition, a severalfold increase in the K-m value was observed by the substitution of the pro-R-p-oxygen of the substrate with sulfur or by the substitution of His(124) of the enzyme with Ala, suggesting that a hydrogen bond is formed between the pro-R-p-oxygen and His(124) These results allow us to propose that the pro-R-p-oxygen contributes to orient His(124) to the best position for the catalytic function through the formation of a hydrogen bond.
• A study on the structure-function relationship of lipopeptide biosurfactants

  M Morikawa, Y Hirata, T Imanaka

  BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR AND CELL BIOLOGY OF LIPIDS 1488 (3) 211 - 218 1388-1981 2000/11 [Refereed][Not invited]
   

  Arthrofactin (AF) and surfactin (SF) are the most effective cyclic lipopeptide biosurfactants ever reported. Linear AF and linear SF were prepared by saponification of lactone ring. The oil displacement activities decreased to one third of their respective original values. When residues of both an aspartic acid and a glutamic acid of SF were methylated or amidated, the activity increased by 20%, although their water solubility was lost. When these amino acid residues were modified by aminomethane sulfonic acid, the activity was drastically decreased probably owing to charge repulsion and structural distortion inhibiting micelle formation. Both AF and SF expressed higher activity under alkaline conditions than acidic conditions. AF was more resistant to acidic conditions than SF and it kept high activity even under pH 0.5. Although SF drastically reduced its activity under acidic conditions, surfactin-Asp/Glu-amido ester and snrfactin-Asp/Glu-methyl ester retained similar activities irrespective of the pH change. A couple of conformers of SF prepared by reverse-phase HPLC showed the same oil displacement activity but different surface tension-reducing activity. AF was produced as a series of different fatty acid chain lengths (from C8 to C12). Among them, AF with fatty acid chain length of C10, which was the main product of the strain, showed the highest activity. (C) 2000 Elsevier Science B.V. All rights reserved.
• Enhancement of the enzymatic activity of ribonuclease HI from Thermus thermophilus HB8 with a suppressor mutation method

  N Hirano, M Haruki, M Morikawa, S Kanaya

  BIOCHEMISTRY 39 (43) 13285 - 13294 0006-2960 2000/10 [Refereed][Not invited]
   

  A genetic method for isolating a mutant enzyme of ribonuclease HI (RNase HI) from Thermus thermophilus HB8 with enhanced activity at moderate temperatures was developed. T. thermophilus RNase HI has an ability to complement the RNase H-dependent temperature-sensitive (ts) growth phenotype of Escherichia coli MIC3001. However, this complementation ability was greatly reduced by replacing Asp(134) which is one of the active site residues, with His, probably due to a reduction in the catalytic activity. Random mutagenesis of the gene encoding the resultant D134H enzyme, followed by screening for second-site revertants, allowed us to isolate three single mutations (Ala(12) --> Ser, Lys(75) --> Met, and Ala(77) --> Pro) that restore the normal complementation ability to the D134H enzyme. These mutations were individually or simultaneously introduced into the wild-type enzyme, and the kinetic parameters of the resultant mutant enzymes for the hydrolysis of a DNA-RNA-DNA/DNA substrate were determined at 30 degreesC. Each mutation increased the k(cat)/K-m value of the wild-type enzyme by 2.1-4.8-fold. The effects of the mutations on the enzymatic activity were roughly cumulative, and the combination of these three mutations increased the k(cat)/K-m value of the wild-type enzyme by 40-fold (5.5-fold in k(cat)). Measurement of thermal stability of the mutant enzymes with circular dichroism spectroscopy in the presence of 1 M guanidine hydrochloride and 1 mM dithiothreitol showed that the T-m value of the triple mutant enzyme, in which all three mutations were combined, was comparable to that of the wild-type enzyme (75.0 vs 77.4 degreesC). These results demonstrate that the activity of a thermophilic enzyme can be improved without a cost of protein stability.
• Identification of the histidine and aspartic acid residues essential for enzymatic activity of a family I.3 lipase by site-directed mutagenesis

  K Hyun-Ju, K Amada, M Haruki, M Morikawa, S Kanaya

  FEBS LETTERS 483 (2-3) 139 - 142 0014-5793 2000/10 [Refereed][Not invited]
   

  A lipase from Pseudomonas sp, MIS38 (PML) is a member of the lipase family 1.3. We analyzed the roles of the five histidine residues (His(30), His(274), His(291), His(313), and His(365)) and five acidic amino acid residues (Glu(253), Asp(255), ASp(262), Asp(275), and Asp(290)), which are fully conserved in the amino acid sequences of family 1.3 lipases, by site-directed mutagenesis, We showed that the mutation of His(313) or Asp(255) to Ala almost fully inactivated the enzyme, whereas the mutations of other residues to Ala did not seriously affect the enzymatic activity. Measurement of the far- and near-UV circular dichroism spectra suggests that inactivation by the mutation of His(313) Or Asp(255) is not due to marked changes in the tertiary structure, We propose that His(313) and Asp(255), together with Ser(207), form a catalytic triad in PML. (C) 2000 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.
• Overproduction in Escherichia coli, purification and characterization of a family I.3 lipase from Pseudomonas sp MIS38

  K Amada, M Haruki, T Imanaka, M Morikawa, S Kanaya

  BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY 1478 (2) 201 - 210 0167-4838 2000/05 [Refereed][Not invited]
   

  Determination of the nucleotide sequence of the gene encoding a lipase from Pseudomonas sp. MIS38 (PML) revealed that PML is a member of the lipase family I.3 and is composed of 617 amino acid residues with a calculated molecular weight of 64 510. Recombinant PML (rPML) was overproduced in Escherichia coli in an insoluble form, solubilized in the presence of 8 M urea, purified in a urea-denatured form and refolded by removing urea in the presence of the Ca2+ ion. Gel filtration chromatography suggests that this refolded protein is monomeric. rPML showed relatively broad substrate specificities and hydrolyzed glyceryl tributyrate and olive oil with comparable efficiencies. rPML was active only in the form of a holoenzyme, in which at least 12 Ca2+ ions bound. These Ca2+ ions bound too tightly to be removed from the protein upon dialysis, but were removed from it upon EDTA treatment. The resultant ape-enzyme was fully active in the presence of 10 mM CaCl2, but was inactive in the absence of the Ca2+ ion. PML has a GXSXG motif, which is conserved in lipases/esterases and generally contains the active-site serine. The mutation of Ser(207) within this motif to Ala completely inactivated PML, suggesting that Ser(207) is the active-site serine of PML. (C) 2000 Elsevier Science B.V. All rights reserved.
• Characterization of ribonuclease HII from Escherichia coli overproduced in a soluble form

  N Ohtani, M Haruki, A Muroya, M Morikawa, S Kanaya

  JOURNAL OF BIOCHEMISTRY 127 (5) 895 - 899 0021-924X 2000/05 [Refereed][Not invited]
   

  Escherichia coli RNase HII is composed of 198 amino acid residues. The enzyme has been overproduced in an insoluble form, purified in a urea-denatured form, and refolded with poor yield [M. Itaya (1990) Proc. Natl. Acad. Sci. USA 87, 8587-8591]. To facilitate the preparation of the enzyme in an amount sufficient for physicochemical studies, we constructed an overproducing strain in which E. coli RNase HII is produced in a soluble form. The enzyme was purified from this strain and its biochemical and physicochemical properties were characterized. The good agreement in the molecular weights estimated from SDS-PAGE (23,000) and gel filtration (22,000) suggests that the enzyme acts as a monomer. From the far-UV circular dichroism spectrum, its helical content was calculated to be 23%. The enzyme showed Mn2+-dependent RNase H activity. Its specific activity determined using H-3-labeled M13 RNA/DNA hybrid as a substrate was comparable to but slightly higher than that of the refolded enzyme, indicating that the enzyme overproduced and purified in a soluble form is more suitable for structural and functional analyses than the refolded enzyme.
• Thermophilic bacteria isolated from a subterranean petroleum reservoir.

  M. Morikawa, T. Kato, M. Haruki, T. Imanaka, S. Kanaya

  In Gratama Workshop 2000 conference report 202 - 203 2000 [Refereed][Not invited]
  
• Decarbonylase, an enzyme which transforms aldehyde to alkene.

  H. Takagi, M. Haruki, N. Murakami, T. Imanaka, M. Morikawa, S. Kanaya

  In Proceedings of Fifth International Symposium on Environmental Biotechnology 2000 [Refereed][Not invited]
  
• Aldehyde dehydrogenase and esterase from a petroleum-degrading bacterium HD-1.

  S. Kanaya, N. Okibe, S. Mizuguchi, M. Haruki, T. Imanaka, M. Morikawa

  In Proceedings of Fifth International Symposium on Environmental Biotechnology 2000 [Refereed][Not invited]
  
• An extremely thermophile which degrades petroleum under high termperature conditions.

  T. Kato, A. Miyanaga, M. Haruki, T. Imanaka, M. Morikawa, S. Kanaya

  In Proceedings of Fifth International Symposium on Environmental Biotechnology 2000 [Refereed][Not invited]
  
• Enzymes from a psychrophilic Shewanella sp. SIB1.

  M. Haruki, N. Ohtani, K. Miura, Y. Suzuki, T. Kato, M. Morikawa, S. Kanaya

  In Proceedings of Fifth International Symposium on Environmental Biotechnology 2000 [Refereed][Not invited]
  
• Petroleum Degradation by an extremely thermophilic bacteria.

  M. Morikawa, T. Kato, A. Miyanaga, M. Haruki, T. Imanaka, S. Kanaya

  In Proceedings of Fifth International Symposium on Environmental Biotechnology 2000 [Refereed][Not invited]
  
• 新規抗菌物質の探索

  森川正章, 金谷茂則, 藤本善久

  大阪大学先端科学技術共同研究センター年報 (6) 18 - 20 2000 [Refereed][Not invited]
  
• 微生物にもとめる火事場の．．．？

  森川 正章

  The News of Engineering. Osaka University. 9 2000 [Refereed][Not invited]
  
• Crystallization and preliminary X-ray study of Pk-REC from a hyperthermophilic archaeon, Pyrococcus kodakaraensis KOD1

  Kazuaki Harata, Noriyuki Ishii, Naeem Rashid, Masaaki Morikawa, Tadayuki Imanaka

  Acta Crystallographica Section D: Biological Crystallography 56 (5) 648 - 649 0907-4449 2000 [Refereed][Not invited]
   

  Pk-REC is a protein which binds to DNA and catalyzes the central step of recombination and repair. The protein was crystallized using the hanging-drop vapour-diffusion method with PEG as a precipitant. Two orthorhombic crystal forms I and II with the same space group P212121 were obtained at pH 8.0 using PEG 3000 and PEG 550 monomethylether, respectively. The unit-cell parameters were a = 151, b = 174, c = 241 Å for form I and a = 151, b = 176, c = 300 Å for form II, indicating that the asymmetric unit contains more than 20 molecules.
• Identification of the gene encoding esterase, a homolog of hormone-sensitive lipase, from an oil-degrading bacterium, strain HD-1

  S Mizuguchi, K Amada, M Haruki, T Imanaka, M Morikawa, S Kanaya

  JOURNAL OF BIOCHEMISTRY 126 (4) 731 - 737 0021-924X 1999/10 [Refereed][Not invited]
   

  The gene encoding an esterase (HDE) was cloned from an oil-degrading bacterium, strain HD-I. HDE is a member of the hormone-sensitive lipase family and composed of 317 amino acid residues with a molecular weight of 33,633, The HDE-encoding gene was expressed in Escherichia coli, and the recombinant protein was purified and characterized. Amino acid sequence analysis indicated that the methionine residue was removed from its NH2-terminus, The good agreement of the molecular weights estimated by SDS-PAGE (35,000) and gel filtration (38,000) suggests that it acts in a monomeric form. HDE showed hydrolytic activity towards p-nitrophenyl esters of fatty acids with an acyl chain length of 2 to 14 and tributyrin, whereas it showed little hydrolytic activity towards p-nitrophenyl oleate (C-18), tricaprylin and triolein, Determination of the kinetic parameters for the hydrolyses of the p-nitrophenyl substrates from C-2 to C-14 indicated that HDE shows a relatively broad substrate specificity. However, comparison of the k(cat)/K-m values indicated that the C-10-C-14 substrates are the most preferred ones, Such a preference for substrates with long acyl chains may be a characteristic of HDE.
• Isolation of TBP-interacting protein (TIP) from a hyperthermophilic archaeon that inhibits the binding of TBP to TATA-DNA

  T Matsuda, M Morikawa, M Haruki, H Higashibata, T Imanaka, S Kanaya

  FEBS LETTERS 457 (1) 38 - 42 0014-5793 1999/08 [Refereed][Not invited]
   

  We have isolated TBP (TATA-binding protein)interacting protein (TIP) from cell lysates of a hyperthermophilic archaeon, Pyrococcus kodakaraensis KOD1, by affinity chromatography with TBP-agarose. Based on the internal amino acid sequence information, PCR primers were synthesized and used to amplify the gene encoding this protein (Pk-TIP). Determination of the nucleotide sequence and characterization of the recombinant protein revealed that Pk-TIP is composed of 224 amino acid residues (molecular weight of 25558) and exists in a dimeric form. BIAcore analyses for the interaction between recombinant Pk-TIP and recombinant Pk-TBP indicated that they interact with each other with an equilibrium dissociation constant, K-D, of 1.24-1.46 mu M. A gel mobility shift assay indicated that Pk-TIP inhibited the interaction between Pk-TBP and a TATA-DNA. Pk-TIP may be one of the archaeal factors which negatively regulate transcription. (C) 1999 Federation of European Biochemical Societies.
• Molecular diversities of RNases H

  N Ohtani, M Haruki, M Morikawa, S Kanaya

  JOURNAL OF BIOSCIENCE AND BIOENGINEERING 88 (1) 12 - 19 1389-1723 1999/07 [Refereed][Not invited]
   

  RNase H is an enzyme that specifically cleaves RNA hybridized to DNA. The enzyme is ubiquitously present in various organisms. Single bacterial and eucaryotic tells often contain two RNases H, whereas single archaeal cells contain only one. To determine whether there is a physiologicaI significance in the ubiquity and multiplicity of the enzyme, and whether all enzymes are evolutionarily diverged from a common ancestor, we carried out phylogenetic analyses of the RNase H sequences. In this report, we demonstrated that RNases H are classified into two major families, Type 1 and Type 2 RNases H, of which only the Type 2 enzymes are present in all Living organisms, including bacteria, archaea, and eucaryotes, suggesting that they represent an ancestral form of RNases H. Based on this information, we discuss the evolutionary relationships and possible cellular functions of RNases H.
• Characterization of petroleum-degrading bacteria from oil-contaminated sites in Vietnam

  NQ Huy, S Jin, K Amada, M Haruki, NB Huu, DT Hang, DTC Ha, T Imanaka, M Morikawa, S Kanaya

  JOURNAL OF BIOSCIENCE AND BIOENGINEERING 88 (1) 100 - 102 1389-1723 1999/07 [Refereed][Not invited]
   

  Four petroleum-degrading bacterial strains, 2TN-NB, 6TBX-CL, MVK2-5, and XCK, were isolated from various oil-contaminated sites in Vietnam. Determination of the nucleotide sequence of the gene encoding 16S rRNA allowed 2TN-NB to be identified as Acinetobacter sp, and the other three stains as Pseudomonas sp. Among the four isolates, 2TN-NB was most effective in degrading crude oil: in 1 d, it degraded 95% of the crude oil in the culture medium (5%, v/v). The isolated strains could also degrade a sulfur-containing aromatic hydrocarbon, dibenzothiophene (DBT), with low efficiency. Except for MVK2-5, which degraded crude oil least efficiently, the isolates produced biosurfactants in amounts sufficient for structural analysis. FT-IR measurement suggested that strains 6TBX-CL and XCK produced glycolipid-type biosurfactants while that produced by 2TN-NB was of the polysaccharide type.
• Identification of catalytically essential residues in Escherichia coli esterase by site-directed mutagenesis

  M Haruki, Y Oohashi, S Mizuguchi, Y Matsuo, M Morikawa, S Kanaya

  FEBS LETTERS 454 (3) 262 - 266 0014-5793 1999/07 [Refereed][Not invited]
   

  Escherichia coli esterase (EcE) is a member of the hormone-sensitive lipase family. We have analyzed the roles of the conserved residues in this enzyme (His(103), Glu(128), Gly(163), Asp(164), Ser(165), Gly(167), Asp(262), Asp(266) and His(292)) by site-directed mutagenesis. Among them, Gly(163), Asp(164), Ser(165), and Gly(167) are the components of a G-D/E-S-A-G motif. We showed that Ser(165), Asp(262), and His(292) are the active-site residues of the enzyme. We also showed that none of the other residues, except for Asp(164), is critical for the enzymatic activity. The mutation of Asp(164) to Ala dramatically reduced the catalytic efficiency of the enzyme by the factor of 10(4) without seriously affecting the substrate binding. This residue is probably structurally important to make the conformation of the active-site functional. (C) 1999 Federation of European Biochemical Societies.
• A unique DNase activity shares the active site with ATPase activity of the RecA/Rad51 homologue (Pk-REC) from a hyperthermophilic archaeon

  N Rashid, M Morikawa, S Kanaya, H Atomi, T Imanaka

  FEBS LETTERS 445 (1) 111 - 114 0014-5793 1999/02 [Refereed][Not invited]
   

  A RecA/Rad51 homologue from Pyrococcus koda-karaensis KOD1 (Pk-REC) is the smallest protein among various RecA/Rad51 homologues, Nevertheless, Pk-Rec is a super multifunctional protein and shows a deoxyribonuclease activity. This deoxyribonuclease activity was inhibited by 3 mM or more ATP, suggesting that the catalytic centers of the ATPase and deoxyribonuclease activities are overlapped. To examine whether these two enzymatic activities share the same active site, a number of site-directed mutations were introduced into Pk-REC and the ATPase and deoxyribonuclease activities of the mutant proteins were determined. The mutant enzyme in which double mutations Lys-33 to Ala and Thr-34 to Ala were introduced, fully lost both of these activities, indicating that Lys-33 and/or Thr-34 are important for both ATPase and deoxyribonuclease activities. The mutation of Asp-112 to Ale slightly and almost equally reduced both ATPase and deoxyribonuclease activities. In addition, the mutation of Glu-54 to Gib did not seriously affect the ATPase, deoxyribonuclease, and UV tolerant activities. These results strongly suggest that the active sites of the ATPase and deoxyribonuclease activities of Pk-REC are common, It is noted that unlike Glu-96 in Escherichia coli RecA, which has been proposed to be a catalytic residue for the ATPase activity, the corresponding residual Glu-54 in Pk-REC is not involved in the catalytic function of the protein. (C) 1999 Federation of European Biochemical Societies.
• 環境問題を大学で学ぶ

  森川 正章

  栄冠をめざしてSPECIAL．河合塾／全国進学情報センター (特別) 1999 [Refereed][Not invited]
  
• CO2固定細菌を利用した地球環境修復システムの構築

  森川 正章

  生産技術 生産技術振興協会 51 (1) 153 - 157 0387-2211 1999 [Refereed][Not invited]
  
• Identification of the genes encoding Mn2+-dependent RNase I-III and Mg2+-dependent RNase HIII from Bacillus subtilis: Classification of RNases H into three families

  N Ohtani, M Haruki, M Morikawa, RJ Crouch, M Itaya, S Kanaya

  BIOCHEMISTRY 38 (2) 605 - 618 0006-2960 1999/01 [Refereed][Not invited]
   

  Database searches indicated that the genome of Bacillus subtilis contains three different genes encoding RNase H homologues. The ypdQ gene encodes an RNase HI homologue with 132 amino acid residues, whereas the rnh and ysgB genes encode RNase HII homologues with 255 and 313 amino acid residues, respectively. RNases HI and HII show no significant sequence similarity. These genes were individually expressed in Escherichia coli; the recombinant proteins were purified, and their enzymatic properties were compared with those of E. coli RNases HI and HII. We found that the ypdQ gene product showed no RNase H activity. The 2.2 kb pair genomic DNA containing this gene did not suppress the RNase H deficiency of an E, coli rnhA mutant, indicating that this gene product shows no RNase H activity in vivo as well. In contrast, the rnh (rnhB) gene product (RNase HII) showed a preference for Mn2+, as did E. coli RNase HII, whereas the ysgB (mhC) gene product (RNase HIII) exhibited a Mg2+-dependent RNase H activity. Oligomeric substrates digested with these enzymes indicate similar recognition of these substrates by B. subtilis and E. coli RNases HII. Likewise, B. subtilis RNase HIII and E. coli RNase HI have generated similar products. These results suggest that B. subtilis RNases HII and HIII may be functionally similar to E, coli RNases HII and HI, respectively. We propose that Mn2+-dependent RNase HII is universally present in various organisms and Mg2+-dependent RNase HIII, which may have evolved from RNase HII, functions as a substitute for RNase HI.
• Gene cloning and characterization of aldehyde dehydrogenase from a petroleum-degrading bacterium, strain HD-1

  Naoko Okibe, Kei Amada, Shin Ichi Hirano, Mitsuru Haruki, Tadayuki Imanaka, Masaaki Morikawa, Shigenori Kanaya

  Journal of Bioscience and Bioengineering 88 (1) 7 - 11 1389-1723 1999 [Refereed][Not invited]
   

  The hd-ald gene encoding aldehyde dehydrogenase (hd-ALDH) from an mixotrophic petroleum-degrading bacterium, strain HD-1 was cloned and sequenced, hd-ALDH (506 amino acids) is a member of the NAD+-dependent aldehyde dehydrogenase group. The hd-ald gene was expressed in Escherichia coli, and the recombinant enzyme was purified and characterized biochemically and enzymatically. The molecular weight of the enzyme was estimated to be 55,000 by SDS-PAGE, and 224,000 by gel filtration chromatography, suggesting that it acts as a tetramer. The CD spectrum suggests that the helical content of the enzyme is 10%. hd-ALDH was active on various aliphatic aldehyde substrates. The K(m) values of the enzyme were 6.4 μM for acetaldehyde, 4.2 μM for hexanal, 2.8 μM for octanal, and 0.84 μM for decanal, whereas the k(cat) values for these substrates were nearly equal (51-64 min-1). These results indicate that hd-ALDH acts preferentially on long-chain aliphatic aldehydes.
• Thermostable glycerol kinase from a hyperthermophilic archaeon: gene cloning and characterization of the recombinant enzyme

  Y Koga, M Morikawa, M Haruki, H Nakamura, T Imanaka, S Kanaya

  PROTEIN ENGINEERING 11 (12) 1219 - 1227 0269-2139 1998/12 [Refereed][Not invited]
   

  The Pk-glpK gene, which encodes glycerol kinase (GK) from a hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1, was cloned and expressed in Escherichia coli, The amino acid sequence of this enzyme (Pk-GK) deduced from the nucleotide sequence showed 57% identity with that of E.coli GK and 37% identity with that of human GK, Pk-GK, which has a molecular weight of 55 902 (497 amino acid residues), was purified from E.coli and characterized, Despite the high sequence similarity, Pk-GK and E.coli GK are greatly divergent in structure and function from each other. Unlike E.coli GK, which exists as a tetramer, Pk-GK exists as a dimer, The preferred divalent cation for Pk-GK is Co2+, instead of Mg2+. The optimum pH and temperature for Pk-GK activity are 8.0 and 80 degrees C, respectively, Pk-GK call utilize other nucleoside triphosphates than ATP as a phosphoryl donor, It is fairly resistant to an allosteric inhibitor of E.coli GK, fructose-1,6-bisphosphate. Determination of the kinetic parameters indicates that the K-m value of the enzyme is 15.4 mu M for ATP and 111 mu M for glycerol and its k(cat) value is 930 s(-1). The enzyme was shown to be fairly resistant to irreversible heat inactivation and still retained 50% of its enzymatic activity even after heating at 100 degrees C for 30 min. Construction of a model for the three-dimensional structure of the enzyme suggests that the formation of extensive ion-pair networks is responsible for the high stability of this enzyme.
• Gene cloning and characterization of recombinant RNase HII from a hyperthermophilic archaeon

  M Haruki, K Hayashi, T Kochi, A Muroya, Y Koga, M Morikawa, T Imanaka, S Kanaya

  JOURNAL OF BACTERIOLOGY 180 (23) 6207 - 6214 0021-9193 1998/12 [Refereed][Not invited]
   

  We have cloned the gene encoding RNase HII (RNase HIIPk) from the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 by screening of a library for clones that suppressed the temperature-sensitive growth phenotype of an mh mutant strain of Escherichia coli. This gene was expressed in an mh mutant strain off. call, the recombinant enzyme was purified, and its biochemical properties were compared with those of E. coli RNases HI and HII, RNase HIIPk is composed of 228 amino acid residues (molecular weight, 25,799) and acts as a monomer, Its amino acid sequence showed little similarity to those of enzymes that are members of the RNase HI family of proteins but showed 40, 31, and 25% identities to those of Methanococcus jannaschii, Saccharomyces cerevisiae, and E. coli RNase HII proteins, respectively. The enzymatic activity,vas determined at 30 degrees C and pH 8.0 by use of an M13 DNA-RNA hybrid as a substrate. Under these conditions, the most preferred metal ions were Co2+ for RNase HIIPk, Mn2+ for E. coli RNase HII, and Mg2+ for E. coli RNase HI. The specific activity of RNase HIIPk determined in the presence of the most preferred metal ion was 6.8-fold higher than that off. coli RNase HII and 4.5-fold lower than that off. coli RNase HI, Like E. coli RNase HI, RNase HIIPk and E. call RNase HII cleave the RNA strand of an RNA-DNA hybrid endonucleolytically at the P-O3' bond. In addition, these enzymes cleave oligomeric substrates in a similar manner. These results suggest that RNase HIIPk and E. coli RNases HI and HII are structurally and functionally related to one another.
• Cloning of a ribonuclease HII gene from a hyperthermophilic archaeon: Purification and characterization of a recombinant enzyme.

  Haruki, M, Hayashi, K, Kouchi, T, Muroya, A, Koga, Y, Morikawa, M, Imanaka, T, Kanaya, S

  J. Bacteriol. 1998/12 [Refereed][Not invited]
  
• Stabilization of ribonuclease HI from Thermus thermophilus HB8 by the spontaneous formation of an intramolecular disulfide bond

  N Hirano, M Haruki, M Morikawa, S Kanaya

  BIOCHEMISTRY 37 (36) 12640 - 12648 0006-2960 1998/09 [Refereed][Not invited]
   

  TO identify factors that contribute to the thermal stability of ribonuclease HI (RNase HI) from Thermus thermophilus HB8, protein variants with a series of carboxyl-terminal truncations and Cys Ala mutations were constructed, and their thermal denaturations were analyzed by CD. The results indicate that Cys(41) and Cys(149) contribute to the protein stability, probably through the formation of a disulfide bond. Peptide mapping analysis for the mutant protein with only two cysteine residues, at positions 41 and 149, indicated that this disulfide bond is partially formed in a protein purified from Escherichia coli in the absence of a reducing reagent but is fully formed in a thermally denatured protein. These results suggest that the thermal stability of T. thermophilus RNase HI, determined in the absence of a reducing reagent, reflects that of an oxidized form of the protein. Comparison of the thermal stabilities and the enzymatic activities of the wild-type and truncated proteins, determined in the presence and absence of a reducing reagent, indicates that the formation of this disulfide bond increases the thermal stability of the protein by 6-7 degrees C in T-m and similar to 3 kcal/mol in Delta G without seriously affecting the enzymatic activity. Since T. thermophilus RNase HT is present in a reducing environment in cells, this disulfide bond probably is not formed in vivo but is spontaneously formed in vitro in the absence of a reducing reagent.
• Production of alkane and alkene from CO2 by a petroleum-degrading bacterium strain HD-1

  M Morikawa, T Iwasa, K Nagahisa, S Yanagida, T Imanaka

  ADVANCES IN CHEMICAL CONVERSIONS FOR MITIGATING CARBON DIOXIDE 114 467 - 470 0167-2991 1998 [Refereed][Not invited]
  
• 炭酸ガスからの石油生成

  森川正章, 金 沙, 金谷茂則, 今中忠行

  農芸化学会誌 72 (4) 532 - 534 0002-1407 1998 [Refereed][Not invited]
  
• 大学推進型新プロジェクトが創る夢

  森川 正章

  大阪大学工業会誌テクノネット 501 9  1998 [Refereed][Not invited]
  
• Production of alkane and alkene from CO2 by a petroleum-degrading bacterium

  M Morikawa, T Iwasa, S Yanagida, T Imanaka

  JOURNAL OF FERMENTATION AND BIOENGINEERING 85 (2) 243 - 245 0922-338X 1998 [Refereed][Not invited]
   

  A petroleum-degrading bacterium, strain HD-1, was grown anaerobically with CO2 and H-2 as Sole carbon and energy sources, respectively. Electron microscopic observation revealed that many granules were accumulated inside the cell. GC/MS analysis of the cell components showed that the bacterium fixed CO2 and produced alkanes/alkenes with carbon numbers from 14 to 30 besides fatty acids and poly-beta-hydroxyalkanoic acids. By employing HD-1 cell extract, fatty acid and fatty aldehyde were found to be utilized as substrates in the alkane/alkene biosynthetic pathways.
• Characterization of a RecA/RAD51 homologue from the hyperthermophilic archaeon Pyrococcus sp KOD1

  N Rashid, M Morikawa, K Nagahisa, S Kanaya, T Imanaka

  NUCLEIC ACIDS RESEARCH 25 (4) 719 - 726 0305-1048 1997/02 [Refereed][Not invited]
   

  The Pk-rec gene, encoding a RecA/RAD51 homologue from the hyperthermophilic archaeon Pyrococcus sp. KOD1, was expressed in Escherichia coli, The recombinant Pk-REC was purified to homogeneity and was shown to be in a dimeric form. A striking property of the purified recombinant Pk-REC was the unusual DNase activity on both single- and double-stranded DNAs along with the ATPase activity, The reaction product of this DNase activity was mononucleotides, The optimum temperature and pH for the DNase activity were 60 degrees C and 8-8.5, respectively. In addition, the metal ion requirement for DNase activity was different from that for the ATPase activity. The protein exhibited no DNase activity in the presence of Zn2+ ion, which was one of the most preferable divalent cations for ATPase activity. Another unique characteristic of the recombinant protein was that the reaction product of ATPase activity was AMP instead of ADP. Pk-REC may represent a common prototype of the RecA family proteins with high RecA-like activity.
• A new type of petroleum-degrading/producing bacterium HD-1.

  M. Morikawa, T. Imanaka

  In Proceedings of International Workshop on Ultra-Long-Term Cryogenic 51 - 60 1997 [Refereed][Not invited]
  
• Gene cloning and characterization of recombinant ribonuclease HII from a hyperthermophilic archaeon.

  Haruki M, Hayashi K, Kochi T, Muroya A, Koga Y, Morikawa M, Imanaka T, Kanaya S

  Annual reports of ICBiotech. 20 803 - 821 1997 [Refereed][Not invited]
  
• 超好熱菌の新機能解析と耐熱酵素に関する研究

  高木昌宏, 金谷茂則, 森川正章, 春木 満, 藤原伸介, 橘 佳永

  大阪大学先端科学共同研究センター年報 (2) 35 - 38 1997 [Refereed][Not invited]
  
• Gene cloning and characterization of recombinant ribose phosphate pyrophosphokinase from a hyperthermophilic archaeon

  N Rashid, M Morikawa, T Imanaka

  JOURNAL OF FERMENTATION AND BIOENGINEERING 83 (5) 412 - 418 0922-338X 1997 [Refereed][Not invited]
   

  The gene encoding ribose phosphate pyrophosphokinase (Pk-RPPK) from a hyperthermophilic archaeon, Pyrococcus sp. KOD1 (Pk), was cloned, sequenced and expressed in Escherichia coli. The recombinant Pk-RPPK (280 aa, 31,113 Da) was purified to homogeneity. The optimal temperature and pH for the enzymatic activity are 50 degrees C and 7.0, respectively. The half-life of the enzymatic activity is 55 min at 70 degrees C. A unique characteristic of the enzyme is that it can utilize CTP and GTP as substrates In addition to ATP. It was also found that Co2+ in particular and Ni2+ markedly enhance the specific activity of the enzyme, as does Mg2+ in the presence of Co2+ (182 units/mg protein).
• Anaerobic degradation and production of alkane/alkene by a new facultative chemoautotrophic bacterium strain HD-1

  T Imanaka, M Morikawa

  GLOBAL ENVIRONMENTAL BIOTECHNOLOGY 401 - 414 1997 [Refereed][Not invited]
  
• Isolation and characterisation of a new lipopeptide biosurfactant produced by Arthrobacter sp MIS38

  M Morikawa, T Imanaka

  GLOBAL ENVIRONMENTAL BIOTECHNOLOGY 585 - 596 1997 [Refereed][Not invited]
  
• A RecA/RAD51 homologue from a hyperthermophilic archaeon retains the major RecA domain only

  N Rashid, M Morikawa, T Imanaka

  MOLECULAR & GENERAL GENETICS 253 (3) 397 - 400 0026-8925 1996/12 [Refereed][Not invited]
   

  A gene encoding a RecA/RAD51 homologue from a hyperthermophilic archaeon, Pyrococcus sp. KOD1 (Pk), was cloned, sequenced and expressed in Escherichia coli. The deduced 210-amino acid sequence was compared to homologues from bacteria (RecA), eukaryotes (RAD51, DMC1) and archaea (RadA). The entire protein from Pk (Pk-REC) basically corresponds to the essential central domain of its counterparts and lacks the two smaller RecA subdomains at the N- and C-termini. The sequence comparison suggests that Pk-REC represents a common prototype of RecA, RAD51, DMC1 and RadA, with higher enzymatic activity. Recombinant Pk-REC was fully active and complemented the ultraviolet light sensitivity of an E. coli recA mutant strain.
• A new mixotrophic bacterium that can fix CO2 and assimilate aliphatic and aromatic hydrocarbons anaerobically

  T Imanaka, M Morikawa

  MOLECULAR BIOLOGY OF PSEUDOMONADS 289 - 297 1996 [Refereed][Not invited]
  
• RecA/RAD51 homologue from a hyperthermophilic archaeon maintains the major domain only.

  Rashid N, Morikawa M, Imanaka T

  Annual reports of ICBiotech. 19 713 - 718 1996 [Refereed][Not invited]
  
• 微生物が人類と地球を救う

  森川正章, 今中忠行

  生産技術 生産技術振興協会 48 (4) 59 - 62 0387-2211 1996 [Refereed][Not invited]
  
• Biological oxidation of alkane to alkene under anaerobic conditions

  M Morikawa, M Kanemoto, T Imanaka

  JOURNAL OF FERMENTATION AND BIOENGINEERING 82 (3) 309 - 311 0922-338X 1996 [Refereed][Not invited]
   

  A petroleum assimilating facultative anaerobic bacterium (strain HD-1) was isolated from an oil field in Shizuoka. The cells were anaerobically grown on CO2 as the sole carbon source in the presence of H-2, and the growth was markedly enhanced by the addition of aromatic or aliphatic hydrocarbon [Morikawa, M, and Imanaka, T.: J. Ferment. Bioeng., 76, 280-283, 1993]. The strain degraded tetradecane (C14) under anaerobic conditions and one of the major metabolic intermediates was identified as 1-dodecene (C12). This result demonstrates that alkanes are biodegradable in the absence of molecular oxygen via a pathway different from that of beta-oxidation. The strain was found to grow on CO2 in the presence of tetradecane and absence of H-2 gas and Light. These findings strongly indicate that the bacterium is capable of anaerobically utilizing alkane as an energy source.
• Isolation of a new mixotrophic bacterium which can fix CO2 and assimilate aliphatic and aromatic hydrocarbons anaerobically

  T Imanaka, M Morikawa

  ENVIRONMENTAL BIOTECHNOLOGY 16 - 27 1996 [Refereed][Not invited]
   

  We isolated a new type of Gram-negative mixotrophic bacterium, Pseudomonas sp. HD-1, which is a facultative anaerobe. Cells were grown on CO2 as a sole carbon source in the presence of H-2. Anaerobic growth of the strain was enhanced by the addition of aliphatic as well as aromatic hydrocarbons. When cells were grown on n-alkane anaerobically, many oil vacuoles formed in the cells and about 40-50% of the dry cell weight was lipid and oil. The total increase in the hydrophilic components of the cells indicated the anaerobic assimilation of,aliphatic hydrocarbon. Electron microscopic observation showed that the cell surface was highly wavy and that the membrane had a multi-layer structure irrespective of the presence of oil. The strain can fix CO2 and produce n-alkane (C-10 to C-20) in the absence of oil. The strain has the following potentials from the viewpoint of bioremediation. 1) Dissolved the oil pollution problem by anaerobic degradation of hydrocarbons, 2) Dissolve the greenhouse effect by CO2 fixation, and 3) Microbial production of oil.
• AN ABNORMALLY ACIDIC TATA-BINDING PROTEIN FROM A HYPERTHERMOPHILIC ARCHAEON

  N RASHID, M MORIKAWA, T IMANAKA

  GENE 166 (1) 139 - 143 0378-1119 1995/12 [Refereed][Not invited]
   

  The gene encoding the TATA-binding protein (PkTBP) from a hyperthermophilic archaeon, Pyrococcus sp, KOD1 (Pk), was cloned and sequenced. An open reading frame with homology to the conserved C-terminal core region of eukaryotic TBP was expressed in Escherichia coli. Specific DNA-binding activity of the recombinant PkTBP (190 amino acids, 21.36 kDa) was also demonstrated, Although it was composed of a structurally direct repeat sequence which is similar to eukaryotic TBP, the total net charge of archaeal TBP was amazingly negative (calculated isoelectric point (pI) was 4.66 and experimentally estimated pi was 4.8). A series of five Glu residues was found at the C terminus of archaeal TBP. These data strongly suggest that a positively charged protein is also involved in the transcription initiation event which might stabilize the structure of the genomic DNA under high-growth-temperature conditions.
• バイオサーファクタント実用化の試み

  今中忠行, 平田善彦, 森川正章

  Rep. Chem. Mat. R & D Found. 10 103 - 106 1995 [Refereed][Not invited]
  
• 石油をつくる細菌発見

  森川正章, 今中忠行

  高圧ガス 高圧ガス保安協会 32 (4) 336 - 339 0452-2311 1995 [Refereed][Not invited]
  
• 石油を作る細菌

  今中忠行, 森川正章

  燃料及燃焼 燃料及燃焼社 62 (5) 323 - 330 0369-3783 1995 [Refereed][Not invited]
  
• 超好熱菌由来チオールプロテアーゼ耐熱性獲得機構の解析

  森川正章

  日本農芸化学会誌 69 (6) 756 - 757 0002-1407 1995 [Refereed][Not invited]
  
• 微生物による石油生産-脂肪族炭化水素の生合成経路について

  森川正章, 今中忠行

  蛋白質 核酸 酵素 共立出版 40 (1) 52 - 60 0039-9450 1995 [Refereed][Not invited]
  
• GENE CLONING AND CHARACTERIZATION OF THERMOSTABLE PEPTIDYL-PROLYL CIS-TRANS ISOMERASE (PPIASE) FROM BACILLUS-STEAROTHERMOPHILUS SIC1

  DJ KIM, M MORIKAWA, M TAKAGI, T IMANAKA

  JOURNAL OF FERMENTATION AND BIOENGINEERING 79 (2) 87 - 94 0922-338X 1995 [Refereed][Not invited]
   

  The peptidyl prolyl cis-trans isomerase (PPIase, EC 5.2.1.8) gene (ppiT) from Bacillus stearothermophilus SIC1 was cloned on the basis of a partial amino acid sequence of the purified enzyme. ppiT was found as an open reading frame (501 bases) which coded for a protein consisting of 167 amino acid residues (molecular weight, 18,349) (GenBank accession number D42050). The cloned ppiT was overexpressed in Eschelichia coil cells using pET-8c as an expression vector. The enzyme was purified by heat treatment and column chromatography on DEAE-Sepharose CL-6B. Purification was about 148-fold and the molecular weight of the enzyme was estimated to be about 18.0 kDa by SDS-PAGE. PPIase activity was determined using synthetic peptide as a substrate in a 2-step reaction coupled with chymotrypsin treatment. The enzyme was stable at pH 7.5-8.0. No heat denaturation was observed when the enzyme was treated at 60 degrees C for 30 min. The PPIase purified from recombinant E. coil has almost the same characteristics as that from B. stearothemophilus SIC1. In refolding solution, the PPIase enhanced the Isomerization rate of unfolded RNase T1.
• PURIFICATION AND CHARACTERIZATION OF A THERMOSTABLE THIOL PROTEASE FROM A NEWLY ISOLATED HYPERTHERMOPHILIC PYROCOCCUS SP

  M MORIKAWA, Y IZAWA, N RASHID, T HOAKI, T IMANAKA

  APPLIED AND ENVIRONMENTAL MICROBIOLOGY 60 (12) 4559 - 4566 0099-2240 1994/12 [Refereed][Not invited]
   

  A hyperthermophilic archaeon strain, KOD1, was isolated from a solfatara at a wharf on Kodakara Island, Kagoshima, Japan. The growth temperature of the strain ranged from 65 to 100 degrees C, and the optimal temperature was 95 degrees C. The anaerobic strain was an S-0-dependent heterotroph. Cells were irregular cocci and were highly motile with several polar flagella. The membrane lipid was of the ether type, and the GC content of the DNA was estimated to be 38 mol%. The 16S rRNA sequence was 95% homologous to that of Pyrococcus abyssi. The optimum growth pH and NaCl concentration of the strain KOD1 were 7.0 and 3%, respectively. Therefore, strain KOD1 was identified as a Pyrococcus sp. Strain KOD1 produced at least three extracellular proteases. One of the most thermostable proteases was purified 21-fold, and the molecular size was determined to be 44 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 45 kDa by gel filtration chromatography. The specific activity of the purified protease was 2,160 U/mg of protein. The enzyme exhibited its maximum activity at approximately pH 7.0 and at a temperature of 110 degrees C, with azocasein as a substrate. The enzyme activity was completely retained after heat treatment at 90 degrees C for 2 h, and the half-life of enzymatic activity at 100 degrees C was 60 min. The proteolytic activity was significantly inhibited by p-chloromercuribenzoic acid or E-64 but not by EDTA or phenylmethylsulfonyl fluoride. Proteolytic activity was enhanced threefold in the presence of 8 mM cysteine. These experimental results indicated that the enzyme was a thermostable thiol protease.
• CONSTRUCTION OF A NEW HOST-VECTOR SYSTEM IN ARTHROBACTER SP AND CLONING OF THE LIPASE GENE

  M MORIKAWA, H DAIDO, S PONGPOBPIBOOL, T IMANAKA

  APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 42 (2-3) 300 - 303 0175-7598 1994/11 [Refereed][Not invited]
   

  Arthrobacter sp. strain MIS38 was transformed with a shuttle vector containing the kanamycin resistant gene kan (derived from Tn5) by an electroporation method. This shuttle vector is from Brevibacterium lactofermentum and Escherichia coli, pULRS8. The following optimal condition of electroporation was determined. A square wave pulse of 1 kV/cm electric field strength for 0.5 ms duration yielded 3 x 10(5) transformants/mu g plasmid DNA. The number of transformants increased with the amount of DNA over the range 0.01-5 mu g. This host-vector system was then used successfully to clone and express a lipase gene from Arthrobacter sp. strain MIS38 into both Arthrobacter sp. MIS38 and E. coli JM109.
• CLONING OF THE AAPT GENE AND CHARACTERIZATION OF ITS PRODUCT, ALPHA-AMYLASE-PULLULANASE (AAPT), FROM THERMOPHILIC AND ALKALIPHILIC BACILLUS SP STRAIN XAL601

  SP LEE, M MORIKAWA, M TAKAGI, T IMANAKA

  APPLIED AND ENVIRONMENTAL MICROBIOLOGY 60 (10) 3764 - 3773 0099-2240 1994/10 [Refereed][Not invited]
   

  A thermophilic and alkaliphilic Bacillus sp. strain, XAL601, was isolated from soil. It produces a thermostable and alkaline-stable enzyme with both alpha-amylase and pullulanase activities. The alpha-amylase-pullulanase gene (aapT) from this Bacillus strain was cloned, and its nucleotide sequence was determined (GenBank accession number D28467). A very large open reading frame composed of 6,096 bases, which encodes 2,032 amino acid residues with an M(r) of 224,992, was found. The deduced amino acid sequence revealed that the four highly conserved regions that are common among amylolytic enzymes were well conserved. These include an active center and common substrate-binding sites of various amylases. In the C-terminal region, a six-amino-acid sequence (Gly-Ser-Gly-Thr-Thr-Pro) is repeated 12 times. The aapT gene was then subcloned in Escherichia coli and overexpressed under the control of the lac promoter. Purification of AapT from this recombinant E. coli was performed, and it was shown that the aapT gene product exhibits both or-amylase and pullulanase activities with one active site. The optimum temperature and pH for enzyme activity were found to be 70 degrees C and pH 9, respectively. Furthermore, AapT was found to strongly adsorb to crystalline cellulose (Avicel) and raw corn starch. Final hydrolyzed products from soluble starch range from maltose (G2) to maltotetraose (G4). Only maltotriose (G3) was produced from pullulan. The enzyme also hgdrolyzes raw starch under a broad range of conditions (60 to 70 degrees C and pH 8 to 9).
• Cloning and analysis of 16S-rRNA gene and transcription factor (TF) IID gene from a hyperthermophilic archaeon strain KOD1.

  Rashid N, Morikawa M, Imanaka T

  Annual reports of ICBiotech. 17 255 - 270 1994 [Refereed][Not invited]
  
• 超好熱始原菌 KOD1 株チオール型プロテアーゼ(TT プロテアーゼ) 遺伝子の検索

  森川 正章

  アマシャムライフサイエンスHighlight (12月) 1994 [Refereed][Not invited]
  
• 微生物による石油分解と石油生成

  今中忠行, 森川正章

  バイオサイエンスとインダストリー バイオインダストリ-協会 52 (11) 898 - 900 0914-8981 1994 [Refereed][Not invited]
  
• CO2を単一炭素源として生育する石油代謝細菌

  森川正章, 今中忠行

  化学と生物 32 690 - 691 1994 [Refereed][Not invited]
  
• A NEW LIPOPEPTIDE BIOSURFACTANT PRODUCED BY ARTHROBACTER SP STRAIN MIS38

  M MORIKAWA, H DAIDO, T TAKAO, S MURATA, Y SHIMONISHI, T IMANAKA

  JOURNAL OF BACTERIOLOGY 175 (20) 6459 - 6466 0021-9193 1993/10 [Refereed][Not invited]
   

  A biosurfactant termed arthrofactin produced by Arthrobacter species strain MIS38 was purified and chemically characterized as 3-hydroxydecanoyl-D-leucyl-D-asparagyl-D-threonyl-D-leucyl-D-l eucyl-D-seryl-L-leucyl-D-Seryl-L-isoleucyl-L-isoleucyl-L-asparagyl lactone. Surface activity of arthrofactin was examined, with surfactin as a control. Critical micelle concentration values of arthrofactin and surfactin were around 1.0 x 10(-5) M and 7.0 x 10(-5) M at 25-degrees-C, respectively. Arthrofactin was found to be five to seven times more effective than surfactin. The minimum surface tension value of arthrofactin was 24 mN/m at a concentration higher than the critical micelle concentration. According to the oil displacement assay, arthrofactin was a better oil remover than synthetic surfactants, such as Triton X-100 and sodium dodecyl sulfate. Arthrofactin is one of the most effective lipopeptide biosurfactants.
• 真核生物のすぐ隣は超好熱菌

  森川正章, 今中忠行

  バイオサイエンスとインダストリー バイオインダストリ-協会 51 (5) 33 - 37 0914-8981 1993 [Refereed][Not invited]
  
• 石油を分解する細菌

  森川正章, 今中忠行

  化学 48 692 - 693 1993 [Refereed][Not invited]
  
• ISOLATION OF A NEW MIXOTROPHIC BACTERIUM WHICH CAN FIX CO2 AND ASSIMILATE ALIPHATIC AND AROMATIC-HYDROCARBONS ANAEROBICALLY

  M MORIKAWA, T IMANAKA

  JOURNAL OF FERMENTATION AND BIOENGINEERING 76 (4) 280 - 283 0922-338X 1993 [Refereed][Not invited]
   

  We isolated a new type of Gram-negative mixotrophic bacterium, Pseudomonas sp. HD-1, which is a facultative anaerobe. Cells were grown on CO2 as a sole carbon source in the presence of H-2. Anaerobic growth of the strain was enhanced by the addition of aliphatic as well as aromatic hydrocarbons. When cells were grown on n-alkane anaerobically, many oil vacuoles formed in the cells and about 40-50% of the dry cell weight was lipid and oil. The total increase in the hydrophilic components of the cells indicated the anaerobic assimilation of aliphatic hydrocarbon. Electron microscopic observation showed that the cell surface was highly wavy and that the membrane had a multi-layer structure irrespective of the presence of oil.
• ISOLATION OF A NEW SURFACTIN PRODUCER BACILLUS-PUMILUS A-1, AND CLONING AND NUCLEOTIDE-SEQUENCE OF THE REGULATOR GENE, PSF-1

  M MORIKAWA, M ITO, T IMANAKA

  JOURNAL OF FERMENTATION AND BIOENGINEERING 74 (5) 255 - 261 0922-338X 1992 [Refereed][Not invited]
   

  Two bacteria (A-1 and B-1) which exhibited large emulsified halos around their colonies on oil-L-agar plates were isolated. These bacteria produced the same biosurfactant, surfactin. Strain A-1 (Psf+: surfactin producing) was identified as Bacillus pumilus and could be transformed with plasmid DNA using an electroporation method. Several surfactin non-producing (Psf-) mutants were obtained by chemical mutagenesis of B. pumilus A-1. By using them as host cells and pC194 as a vector plasmid, we cloned DNA fragments which complemented Psf- alleles. One of them (2 kb HindIII fragment) could transform Bacillus subtilis MI113 (a derivative of Marburg strain 168 and Psf-) to a Psf+ cell as well. Nucleotide sequence of the 2 kb fragment was determined and three large open reading frames (ORF1, 2, 3) were found. Deletion analysis of the recombinant plasmid indicated that ORF3 (699 bp, 233 amino acid residues) is essential for surfactin production. The gene was designated as psf-1 and a 25 kDa translation product was detected. The nucleotide sequence of psf-1 exhibited high homology with an unknown open reading frame, orfX, upstream to the gramicidin S biosynthesis operon of Bacillus brevis. It is suggested that the deduced gene products of unknown orfX in B. brevis and psf-1 in B. pumilus may share the same function.
• 細胞内タンパク質の選択的分解

  森川正章, 今中忠行

  バイオサイエンスとインダストリー バイオインダストリ-協会 49 (9) 969 - 972 0914-8981 1991 [Refereed][Not invited]
  
• Cloning in Bacillus subtilis of thermostable and alkalophilic amylase from a thermophilic Bacillus sp.

  Ganrong X, Lee S-P, Takagi M, Morikawa M, Imanaka T

  Annual reports of ICBiotech. 13 121 - 128 1990 [Refereed][Not invited]
  
• A STRUCTURAL REQUIREMENT IN THE SUBSITE-F OF LYSOZYME - THE ROLE OF ARGININE-115 IN HUMAN LYSOZYME REVEALED BY SITE-DIRECTED MUTAGENESIS

  M MURAKI, M MORIKAWA, Y JIGAMI, H TANAKA

  EUROPEAN JOURNAL OF BIOCHEMISTRY 179 (3) 573 - 579 0014-2956 1989/02 [Refereed][Not invited]
  
• ENGINEERING OF HUMAN LYSOZYME AS A POLY-ELECTROLYTE BY THE ALTERATION OF MOLECULAR-SURFACE CHARGE

  M MURAKI, M MORIKAWA, Y JIGAMI, H TANAKA

  PROTEIN ENGINEERING 2 (1) 49 - 54 0269-2139 1988/04 [Refereed][Not invited]
  
• THE ROLES OF CONSERVED AROMATIC AMINO-ACID-RESIDUES IN THE ACTIVE-SITE OF HUMAN LYSOZYME - A SITE-SPECIFIC MUTAGENESIS STUDY

  M MURAKI, M MORIKAWA, Y JIGAMI, H TANAKA

  BIOCHIMICA ET BIOPHYSICA ACTA 916 (1) 66 - 75 0006-3002 1987/11 [Refereed][Not invited]
  
• ENGINEERING OF THE ACTIVE-SITE OF HUMAN LYSOZYME - CONVERSION OF ASPARTIC ACID-53 TO GLUTAMIC-ACID AND TYROSINE-63 TO TRYPTOPHAN OR PHENYLALANINE

  M MURAKI, Y JIGAMI, M MORIKAWA, H TANAKA

  BIOCHIMICA ET BIOPHYSICA ACTA 911 (3) 376 - 380 0006-3002 1987/02 [Refereed][Not invited]
  

Books etc

• Remediation by Floating Plants

  Masaaki Morikawa (Single work)
  2023/01
• Bioremediation: From Key Enzymes to Practical Technologies

  Masaaki Morikawa (Single work)
  Springer 2023/01
• Design of Materials and Technologies for Environmental Remediation

  Shunitz Tanaka, Masaaki Kurasaki, Masaaki Morikwa, Yuichi Kamiya (Joint editor)
  Springer 2023/01
• バイオサーファクタント”、第10章低炭素社会への取組み、「応用微生物学 第３版」

  MORIKAWA, Masaaki 
  文永堂出版 2016
• Chap. 12. Comparison of the degradation activity of biofilm-associated versus planktonic cells pp219-232

  Morikawa M, Washio K 
  Caister Academic Press 2016
• Chap. 13A Using microbial biofilms to enhance the phytoremediation of contaminants in soil and water – A trial for sustainable phenol degradation by duckweed-colonizing biofilms pp 233-240

  Morikawa M, Yamaga F, Suzuki K, Kurashina K, Miwa K, Washio K 
  Caister Academic Press 2016
• 植物機能のポテンシャルを活かした環境保全・浄化技術

  森川正章 (Contributor第４章３節 PGPR (Plant Growth Promoting Rhizobacteria) の環境保全・修復への利用 pp 169-176)
  シーエムシー出版 2011
• 微生物増殖学の現在・未来

  森川 正章 (Contributorバイオフィルム―微生物の生存戦略を生かすpp 444-454)
  地人書館 2009
• バイオフィルムの基礎と制御

  森川正章, 鷲尾健司 (Contributor将来展望 バイオフィルムがもたらすブレークスルーの可能性pp 389-399, バイオサーファクタントを利用したバイオフィルムの形成と阻害pp 278-287)
  エヌティーエス 2008
• ナノバイオ大事典

  森川 正章 (Contributorバイオフィルム pp 420-423)
  テクノシステム 2007
• バイオプロセスハンドブック

  森川 正章 (Contributor4章 微生物工学の基礎 pp 111-122)
  エヌティーエス 2007
• 環境修復の科学と技術

  森川 正章 (Contributor第４章３節 バイオレメディエーション pp150-186)
  北海道大学出版会 2007
• 生物学（上）

  森川 正章 (Joint translation１４章)
  培風館 2006
• 食品のストレス環境と微生物

  森川 正章 (Contributor第9章1節．バイオフィルムの生成と生存メカニズム pp 221-228)
  サイエンスフォーラム 2004
• 農芸化学の事典

  森川 正章 (Contributor6.3.2. 直鎖状炭化水素の分解 pp749-751)
  朝倉書店 2003
• 微生物利用の大展開

  森川正章, ロベルト・コルター (Contributor4.2.3. バイオサーファクタント pp 993-1002, 1.3.2. バイオフィルム–微生物たちの街 pp 178-181)
  エヌティーエス 2002
• 生命工学

  森川 正章 (Contributor第６章 微生物工学 pp155-179)
  共立出版 2000
• 生物工学実験書

  森川正章, 高木昌宏, 今中忠行 (Contributor第4章(4.4.2) 第5章(5.4.2))
  培風館 1992

Conference Activities & Talks

• Enhanced Duckweed Biomass Production Utilizing Novel Plant Growth-Promoting Bacteria  [Invited]

  MORIKAWA, Masaaki

  China-Japan Environmental Microbiology Forum 2016, Chongqing, China  2016/11
• 有用微生物の農作物への新しい展開とその将来像  [Invited]

  森川 正章

  日本生物工学会2016年度大会シンポジウム 於、富山国際会議場  2016/09
• Insight into aquatic plant-microbe interaction  [Invited]

  MORIKAWA, Masaaki

  Thai Research Expo, 2016, Bangkok, Thai  2016/08
• 海底メタン生産環境中の共生関係機構の解析  [Invited]

  森川 正章

  日本農芸化学会2016年度大会シンポジウム 於、札幌コンベンションセンター  2016/03
• 微生物伝播の足場“バイオフィルム”  [Invited]

  森川 正章

  日本細菌学会第89回総会シンポジウム 於、大阪国際交流センター  2016/03
• Mutualistic biofilms- benefits for plants and animals  [Invited]

  MORIKAWA, Masaaki

  The 27th Annual Meeting of the Thai Society for Biotechnology and international conference (TSB2015) Bangkok, Thailand  2015/11
• A new plant breeding technology enforcing mutualistic interaction between aquatic plants and their associated bacteria  [Invited]

  MORIKAWA, Masaaki

  International Symposium on Microbial Research and Biotechnology for Biomass Utilization. JR HAKATA CITY, Fukuoka, Japan  2015/11
• 根圏微生物共生系を活用した高次植生バイオプロセスの開発  [Invited]

  森川 正章

  2015年度北日本支部仙台シンポジウム 於、仙台市  2015/09
• 水生植物と表層微生物の新しい共生メカニズム  [Invited]

  森川 正章

  日本農芸化学会2015年度大会シンポジウム 於、岡山大学  2015/03
• Plant growth-promoting bacteria are natural and powerful tools for enhancement of duckweed production  [Invited]

  MORIKAWA, Masaaki

  – Construction of an international research network for duckweed and scheme for the application to water environmental improvement –SPIRITS program Kyoto University Dept. Botany, Grad. Sch. Sci., Kyoto Univ.  2015/03
• 微生物に魅せられて  [Invited]

  森川 正章

  日清オイリオグループ株式会社 研究所内講演会 於、横須賀市  2014/12
• 水生植物の高機能化による革新的植生浄化技術の開発  [Invited]

  森川 正章

  日本水処理生物学会第51回大会 於、甲府市  2014/11
• Isolation and characterization of biosurfactant producing microorganisms from natural resources in Thailand  [Invited]

  MORIKAWA, Masaaki

  New Core to Core Program Advanced Research Networks. Bangkok, Thailand  2014/08
• Development of effective bioremediation technology utilizing beneficial biofilms  [Invited]

  MORIKAWA, Masaaki

  New Core to Core Program Advanced Research Networks. Brawijaya Univesity, Indonesia  2014/08
• 好熱菌の諸特性〜その特異な酵素から生態まで  [Invited]

  森川 正章

  2014年度第一回バイオ単分子研究会 於、伊豆市  2014/07
• Application of Beneficial Plant-Microbe Interaction for Sustainable Industry  [Invited]

  MORIKAWA, Masaaki

  Joint Symposium on Environmental Science 2013 –Bridging Finland and Japan – at University of Helsinki, Helsinki, Finland  2013/11
• バイオフィルム工学による効率的な環境汚染物質分解  [Invited]

  森川 正章

  JSR株式会社社内講演会（四日市市）  2013/09
• 水生植物の根圏強化による省エネ型水質浄化  [Invited]

  森川 正章

  2013年度 環境バイオテクノロジー学会シンポジウム 於、北九州国際会議場（北九州市）  2013/06
• High Temperature Petroleum Reservoir Microbiology  [Invited]

  MORIKAWA, Masaaki

  Plenary Lecture. 42nd Convention Philippine Society for Microbiology, Tagaytay, Philippine  2013/04
• 教育講演 細菌の生存戦略としてのバイオフィルム形成  [Invited]

  森川 正章

  47回緑膿菌感染症研究会  2013/02
• Upgrading the function of aquaplants utilizing non-legume rhizobacteria  [Invited]

  MORIKAWA, Masaaki

  International Workshop for Plant Response to Mineral Stress  2012/12
• Beneficial biofilm formation by environmental bacteria in oil contaminated soils and the rhizosphere of duckweeds  [Invited]

  MORIKAWA, Masaaki

  Microbiology Seminars at University of Helsinki  2012/10
• Could the utilization of biofilms, surface attached microbial communities, lead to a game-change in green biotechnology?  [Invited]

  MORIKAWA, Masaaki

  Lecture of Environmental Biotechnology at Aalto University  2012/10
• Could the utilization of biofilms, surface attached microbial communities, lead to a game-change in green biotechnology?  [Invited]

  MORIKAWA, Masaaki

  Royal Swedish Academy of Engineering Sciences (IVA) Lecture  2012/10
• Acinetobacter calcoaceticus P23のコウキクサの根圏への付着に関する検討  [Not invited]

  蜂谷祥之, QUACH Angela, 尾形有香, 黒田真史, 井上大介, 森川正章, 池道彦

  平成２４年度生物工学会  2012/10
• Beneficial biofilm formation by environmental bacteria in oil contaminated soils and the rhizosphere of duckweeds  [Invited]

  MORIKAWA, Masaaki

  Seminar at Uppsala Biocenter SLU  2012/10
• Could the utilization of biofilms, surface attached microbial communities, lead to a game-change in green biotechnology?  [Invited]

  MORIKAWA, Masaaki

  Lecture at Technical University of Denmark  2012/10
• Cyclic lipopeptide antibiotics as Hsp90 inhibitors  [Not invited]

  H. Nakamoto, Y. Yokoyama, S. Minagawa, Y. Kondoh, K. Sueoka, H. Osada, M. Morikawa

  6th International Conference on the Hsp90 Chaperone Machine  2012/09
• 低温菌Shewanella sp. SIB1由来エステラーゼの取得および特性解析  [Not invited]

  小林隆一, 平野展孝, 森川正章, 金谷茂則, 春木 満

  平成２４年度化学系学協会東北大会  2012/09
• How do bacteria treat with interfaces and surfaces?  [Invited]

  MORIKAWA, Masaaki

  Invited seminar at L'Oréal Aulnay Research Center  2012/09
• Characterization of an extremely thermophilic bacterium, Coprothermobacter sp. PM9-2, isolated from an undersea petroleum reservoir  [Not invited]

  W. Urushibata, T. Funayama, M. Morikawa

  Extremophiles2012  2012/09
• Anammox 細菌Candidatus‘Brocadia sinica’の膜画分に高発現するタンパク質に関する研究  [Invited]

  東條ふゆみ, 伊東義兼, 岡部 聡, 森川正章

  平成２４年度環境バイオテクノロジー学会（学会奨励賞受賞講演）  2012/06
• Nitrosomonas europaeaのアンモニア酸化活性を促進する因子の同定  [Not invited]

  折山徹郎, 坂上景子, 三輪京子, 森川正章

  平成２４年度環境バイオテクノロジー学会  2012/06
• Beneficial biofilm formation by environmental bacteria  [Invited]

  MORIKAWA, Masaaki

  Invited Seminar at Harvard Medical School  2012/06
• Analysis of LadA-related long-chain alkane monooxygenase in an extremely thermophilic Geobacillus thermoleovorans B23  [Not invited]

  C. Boonmak, Y. Takahashi, M. Honma, M. Morikawa

  ASM2012  2012/06
• バイオフィルムの形成〜微生物の優れた生存戦略  [Invited]

  森川 正章

  バイオミメティクス研究会シンポジウム  2011/12
• 海底油田から単離した高度好熱菌Coprothermobacter sp. PM9-2株の諸特性解析  [Not invited]

  漆畑亘, 船山哲, 森川正章

  第12回極限環境生物学会  2011/11
• Enhancement of water purification activity by reconstructing rhizobacterial community  [Invited]

  MORIKAWA, Masaaki

  JST-NSFC “China-Japan Workshop on Novel Remediation Technologies for Wastewater Environment Conservation"  2011/10
• 有益なバイオフィルムの環境技術への利用  [Invited]

  森川 正章

  日本歯科保存学会2011年度秋季学術大会シンポジウム  2011/10
• Analysis of LadA-related long-chain alkane monooxygenase in an extremely thermophilic Geobacillus thermoleovorans B23  [Not invited]

  C. Boonmak, Y. Takahashi, M. Morikawa

  第63回日本生物工学会大会  2011/09
• Marinobacter属細菌と共存する親属海洋細菌の混合バイオフィルム形成  [Not invited]

  森川正章, 栄木悠, 柞木田なつみ, 鷲尾健司

  第63回日本生物工学会大会  2011/09
• Functional characterization of Thermococcus kodakaraensis S-layer protein in E. coli  [Not invited]

  T. Agata, W. Urushibata, T. Kanai, H. Atomi, T. Imanaka, M. Morikawa

  Thermophiles  2011/09
• Analysis of multiple alkB genes from Geobacillus thermoleovorans B23  [Not invited]

  C. Boonmak, Y. Takahashi, M. Morikawa

  IUMS2011  2011/09
• Purification of azoreductase from a bacterium strain that degrades Orange G dye  [Not invited]

  S. Murakami, V. Gurning, Y. Itoh, M. Morikawa

  IUMS2011  2011/09
• Isolation of an aerobic chemoheterotroph that positively affects the activity of ammonia-oxidizing bacteria  [Not invited]

  K. Sakagami, Y. Itoh, M. Matsumoto, M. Morikawa

  IUMS2011  2011/09
• Isolation and characterization of Rhizobium sp. R8 that forms biofilms on a toilet bowl  [Not invited]

  T. Fukano, Y. Takahashi, M. Gomi, Y. Osaki, M. Morikawa

  IUMS2011  2011/09
• A novel thermotolerant ammonia-oxidizing bacterium, Nitrosomonas thermotolerans JPCCT2, was isolated from activated sludge in a thermal power station in Japan  [Not invited]

  Y. Itoh, M. Matsumoto, M. Morikawa

  IUMS2011  2011/09
• Identification of novel plant-growth promoting factor produced by Acinetobacter calcoaceticus P23, a rhizobacterium of Lemna aoukikusa  [Not invited]

  S. Chida, M. Morikawa

  IUMS2011  2011/09
• An extremely thermophilic bacterium, Coprothermobacter sp. PM9-2, isolated from an offshore petroleum reservoir in the South China Sea  [Not invited]

  W. Urushibata, T. Funayama, M. Morikawa

  国際長期生体学研究ネットワーク（ILTER）年次会議2011  2011/09
• 生物界面活性剤と微生物膜  [Invited]

  森川 正章

  室蘭工業大学工学研究科応用理化学系専攻 講演会  2011/07
• Co-beneficial biofilms on the duckweed roots  [Not invited]

  MORIKAWA, Masaaki

  FEMS 2011 4th Congress of European Microbiologists  2011/06
• 原油汚染土壌修復技術へのバイオフィルムの適用  [Not invited]

  森川正章, 高橋康徳

  環境バイオテクノロジー学会2011年度大会  2011/06
• Isolation of a novel thermotolerant ammonia-oxidizing bacterium, Nitrosomonas thermotolerans JPCCT2, from activated sludge in a thermal power station in Japan  [Not invited]

  Y. Itoh, M. Matsumoto, M. Morikawa

  111th ASM General Meeting  2011/05
• Analysis of the granule formation by Anammox bacteria  [Not invited]

  F. Tojo, Y. Itoh, S. Okabe, M. Morikawa

  第一回国際アナモックスシンポジウム  2011/05
• 好熱性アルカン分解細菌 Geobacillus thremoleovorans B23由来 alkB 遺伝子の多様性解析  [Not invited]

  高橋康徳, チャニター ブーンマク, 森川正章

  日本農芸化学会2011年度大会  2011/03
• 耐熱性を有する新規アンモニア酸化細菌の単離ならびに諸性質  [Not invited]

  伊東義兼, 松本光史, 森川正章

  日本農芸化学会2011年度大会  2011/03
• 便器表面初期付着細菌群の菌叢解析とバイオフィルム形成  [Not invited]

  深野透, 高橋康徳, 五味満裕, 大崎幸彦, 森川正章

  日本農芸化学会2011年度大会  2011/03
• 根圏作用を高度利用した次世代型環境浄化技術の開発  [Invited]

  森川 正章

  農芸化学会研究企画賞受賞講演  2011/03
• 難分解性アゾ染料Orange Gを分解する細菌およびその還元酵素の精製  [Not invited]

  村上 峻, グルニング ボルタ, 伊東義兼, 森川正章

  生化学会  2010/12
• 超好熱始原菌Thermococcus kodakaraensis KOD1由来細胞表層タンパク質Slpを発現する細菌の凝集性およびバイオフィルム形成能の評価  [Not invited]

  漆畑 亘, 阿形朋子, 金井 保, 跡見晴幸, 今中忠行, 森川正章

  生化学会  2010/12
• 根圏作用を高度利用した光駆動型バイオ環境浄化技術の開発  [Invited]

  森川 正章

  北海道大学大学院農学研究院寄付分野講演会  2010/12
• ウキクサ根圏細菌を利用した持続的水質浄化技術  [Invited]

  森川 正章

  北海道バイオ産業クラスターフォーラム 技術シーズ公開会  2010/10
• Biofilm formation on the duckweed roots expands substrate specificity of alkane degrading rhizobacteria  [Invited]

  M. Morikawa

  China-Japan Workshop on Novel Remediation Technologies for Wastewater Environment Conservation  2010/10
• The role of urease activity on biofilm and urolith formation by Staphylococcus sp. T-02 that was isolated from a toilet bowl.  [Not invited]

  K. Oki, K. Washio, D. Matsui, Y. Hirata, M. Morikawa

  IBS2010  2010/09
• Origin of Peroxisomal Beta-oxidation Pathway Suggested by Extremely Thermophilic Alkane Degrading Bacteria  [Invited]

  M. Morikawa

  BIT’s 1st Annual World Congress of Petromicrobiology  2010/07
• Enhanced Production of Vitamin K2 by Bacillus subtilis natto Biofilms  [Invited]

  M. Morikawa, K. Yamazaki, H. Aibara, K.Washio

  BIT’s 3rd Annual World Congress of Industrial Biotechnology  2010/07
• 耐熱性を有する新規アンモニア酸化細菌の単離ならびにその諸性質  [Not invited]

  伊東義兼, 坂上景子, 松本光史, 鷲尾健司, 森川正章

  生化学会北海道支部例会  2010/07
• アンモニア酸化細菌のアンモニア酸化活性に影響を与える従属栄養細菌  [Not invited]

  坂上景子, 伊東義兼, 松本光史, 鷲尾健司, 森川正章

  生化学会北海道支部例会  2010/07
• Sustainable biodegradation of phenol by a symbiotic Acinetobacter calcoaceticus P23 that was isolated from the rhizosphere of duckweed Lemna aoukikusa  [Not invited]

  F. Yamaga, K. Washio, M. Morikawa

  110th ASM General Meeting  2010/05
• (p)ppGppの合成・代謝酵素SpoTによる環状リポペプチドの生産制御  [Not invited]

  鷲尾健司, リムシューピン, ルーンサワンニラン, 森川正章

  日本農芸化学会  2010/03
• フロックを形成するアンモニア酸化細菌の単離ならびにその諸性質  [Not invited]

  伊東義兼, 松本光史, 鷲尾健司, 森川正章

  日本農芸化学会  2010/03
• Nitrosomonas europaeaのアンモニア酸化活性に影響を与える従属栄養細菌  [Not invited]

  坂上景子, 伊東義兼, 松本光史, 鷲尾健司, 森川正章

  日本農芸化学会  2010/03
• ウキクサから単離したアルカン分解根圏細菌の評価  [Not invited]

  鈴木和也, 山賀文子, 鷲尾健司, 森川正章

  日本農芸化学会  2010/03
• Pseudomonas 属細菌の環状リポペプチド生産制御機構  [Invited]

  森川 正章

  北海道大学シンポジウム「北大の研究者達がつむぐ微生物の世界」  2009/12
• ウキクサと根圏細菌の相利共生作用による汚染浄化法  [Invited]

  森川正章, 鷲尾健司

  水処理生物学会  2009/11
• Characterization of crude oil degrading rhizobacteria  [Not invited]

  Kazuya SUZUKI

  Summary of 12th HU-SNU Joint Symposium  2009/11
• 超好熱性アーキアThermococcus kodakaraensis KOD1由来細胞表在タンパク質Slpの機能解析  [Not invited]

  阿形朋子, 鷲尾健司, 金井 保, 跡見晴幸, 今中忠行, 森川正章

  生化学会  2009/10
• 火力発電所硝化槽より単離した新規メタノール資化細菌の諸特性解析  [Not invited]

  石川絵里奈, 伊東義兼, 鷲尾健司, 松本光史, 森川正章

  生化学会  2009/10
• Biofilm formation and effective production of menaquinone-7 (Vitamin K2) by Bacillus subtilis: Lost of the world in environmental bacteria.  [Invited]

  Kenji Washio, Masaaki Morikawa

  Workshop Argentina-Japan “Bioscience and Biotechnology for the Promotion of Agriculture and Food Production”  2009/08
• Studies on a lipopeptide biosurfactant, arthrofactin – structure, biosynthesis, transportation  [Invited]

  MORIKAWA, Masaaki

  Invited lecture at UC Berkeley, California,  2009/08
• バイオフィルムに見られるナフタレン分解活性遅延の理由  [Not invited]

  嶋田恒平, 鷲尾健司, 森川正章

  環境バイオテクノロジー学会  2009/06
• 微生物によるジクロロメタン分解に関する研究  [Not invited]

  岩崎一弘, 中杉奈央, 大川恵, 菅田曜, 森川正章, 宮崎英男, 坂間至朗

  環境バイオテクノロジー学会  2009/06
• Genetic analysis of synthetic regulation and transportation of lipopeptide type biosurfactant.  [Invited]

  N. Roongsawang, S. P. Lim, K. Washio, M. Morikawa

  Biosurfactant workshop  2009/04
• 微生物から見た界面の世界—バイオサーファクタントとバイオフィルム  [Invited]

  森川 正章

  日本化粧品技術者会  2009/04
• バイオフィルム利用の将来展望  [Invited]

  森川 正章

  農芸化学会大会  2009/03
• Anammox細菌のグラニュール形成に関するプロテオミック解析  [Not invited]

  東條ふゆみ, 伊東義兼, 鷲尾健司, 岡部聡, 森川正章

  農芸化学会大会  2009/03
• 根圏細菌Acinetobacter sp. P23株のバイオフィルム形成遺伝子の探索  [Not invited]

  羽山亨, 山賀文子, 鷲尾健司, 森川正章

  農芸化学会大会  2009/03
• バイオフィルム形成能の高いアルカン分解細菌の探索  [Not invited]

  高橋康徳, 嶋田恒平, 鷲尾健司, 森川正章

  農芸化学会大会  2009/03
• 枯草菌バイオフィルムによるメナキノンの菌体外生産  [Not invited]

  山崎和彦, 相原悠, 大胡康, 鷲尾健司, 森川正章

  農芸化学会大会  2009/03
• Sustainable Bioremediation Technology by Utilizing Biofilms  [Invited]

  Morikawa, F. Yamaga, K. Shimada, K. Washio

  International symposium on Biotechnological Approaches to Environmental Science for Energy Production and Sustainability.  2009/02
• 環状リポペプチド、アルスロファクチンの産生を制御する (p)ppGpp合成・代謝酵素、SpoTの機能解析  [Not invited]

  鷲尾健司, リムシューピン, ルーンサワンニラン, 森川正章

  日本分子生物学会•日本生化学会合同大会2008  2008/12
• Staphylococcus 属細菌のバイオフィルム形成における尿素分解酵素（ウレアーゼ）の役割  [Not invited]

  大木海平, 松井大悟, 平田善彦, 鷲尾健司, 森川正章

  日本分子生物学会•日本生化学会合同大会2008  2008/12
• バイオフィルム研究の様々な切り口ー分子、細胞、生態ー  [Invited]

  森川 正章

  日本微生物生態学会２０年度大会  2008/11
• Advances in Biofilm Research to Inhibit Biocorrosion  [Invited]

  MORIKAWA, Masaaki

  ARO workshop  2008/09
• 新規アゾ染料分解脱色細菌の探索  [Not invited]

  文 Volta, R. T. Gurning, 鷲尾健司, 森川正章

  環境バイオテクノロジー学会２０年度大会  2008/06
• Efficacy of biofilm formation by naphthalene degrading Pseudomonas stutzeri T102 toward bioremediation technology.  [Not invited]

  ASM 108th  2008/06
• Biofilm formation by Bacillus subtilis 168, incompatible function of sfp  [Not invited]

  H. Aibara, K. Washio, M. Morikawa

  ASM 108th  2008/06
• A lipopeptide biosurfactant produced by Pseudomonas sp. MIS38, characterization and functional analyses of the synthetase gene.  [Invited]

  M. Morikawa

  Invited lecture at East China University of Science and Technology  2008/05
• Efficacy of biofilm formation by naphthalene degrading Pseudomonas stutzeri T102 toward bioremediation technology.  [Invited]

  M. Morikawa, K. Shimada, K. Washio

  iBIO2008  2008/05
• 小便器内壁から単離した細菌のバイオフィルム形成能  [Not invited]

  大木海平, 松井大悟, 平田善彦, 鷲尾健司, 森川正章

  農芸化学会  2008/03
• Pseudomonas stutzeri バイオフィルムのナフタレン分解活性と土壌安定性  [Not invited]

  嶋田恒平, 片岡剛文, 鷲尾健司, 森川正章

  農芸化学会  2008/03
• 超好熱性アーキアThermococcus kodakaraensis KOD1 の浮遊細胞とコロニー細胞を用いたプロテオーム解析  [Not invited]

  阿形朋子, 松見理恵, 鷲尾健司, 金井保, 跡見晴幸, 今中忠行, 森川正章

  農芸化学会  2008/03
• 食中毒原因細菌のバイオフィルムと浮遊細胞の加熱処理耐性比較  [Not invited]

  東條ふゆみ, 佐藤 禅, 鷲尾健司, 森川正章

  農芸化学会  2008/03
• Pseudomonas sp. MIS38株由来の二つの環状リポペプチド類ABC-トランスポーター  [Not invited]

  Siew Ping Lim, Niran Roongsawang, 鷲尾健司, 森川正章

  生化学会  2007/12
• フタル酸エステル分解細菌の単離と諸特性解析  [Not invited]

  菅原寛明, 陳 志銘, 鷲尾健司, 森川正章

  生化学会  2007/12
• Pseudomonas sp. MIS38株の成長相とバイオサーファクタント生産  [Not invited]

  鷲尾健司, Siew-Ping Lim, Roongsawang Niran, 森川正章

  生化学会  2007/12
• バイオフィルムってなんだろう  [Invited]

  森川 正章

  地球環境科学研究院公開講座「快適な環境をまもる微生物のはたらきと姿」  2007/09
• 微生物の生存戦略：バイオフィルムとバイオサーファクタント  [Invited]

  森川 正章

  日清製粉基礎研究所セミナー  2007/07
• 環境微生物の生存戦略ーバイオフィルムー  [Invited]

  森川正章, 相原 悠, 山崎和彦, 鷲尾健司

  生化学会北海道支部シンポジウム  2007/07
• Pseudomonas stutzeri バイオフィルムのナフタレン分解活性と土壌安定性  [Not invited]

  嶋田恒平, 片岡剛文, 鷲尾健司, 森川正章

  環境バイオテクノロジー学会  2007/06
• 細菌バイオフィルムと浮遊細胞の耐熱性比較  [Not invited]

  東條ふゆみ, 佐藤 禅, 鷲尾健司, 森川正章

  環境バイオテクノロジー学会  2007/06
• フタル酸エステル分解細菌の単離と諸特性解析  [Not invited]

  菅原寛明, 陳 志銘, 鷲尾健司, 森川正章

  環境バイオテクノロジー学会  2007/06
• 非リボソーム型ペプチド合成酵素の解析と機能改変  [Invited]

  MORIKAWA, Masaaki

  第8回 酵素応用シンポジウム  2007/06
• Genes Responsible for the Transportation of Arthrofactin (Arf) in Pseudomonas sp. MIS38  [Not invited]

  Lim SP, Roongsawang N, Washio K, Morikawa M

  ASM 107th Meeting  2007/05
• バイオフィルムとバイオサーファクタント  [Invited]

  森川 正章

  日清製粉研究所セミナー  2007/04
• 水生植物からの汚染物質分解能を有する微生物の探索  [Not invited]

  山賀 文子, 竹井 大亮, 鷲尾 健司, 森川 正章

  農芸化学会  2007/03
• 海洋性細菌を用いた複数種バイオフィルム形成能の評価  [Not invited]

  柞木田 なつみ, 岡田 千尋, 鷲尾 健司, 森川 正章

  農芸化学会  2007/03
• 枯草菌バイオフィルム形成に与えるサーファクチン生産の影響  [Not invited]

  相原 悠, 鷲尾 健司, 森川 正章

  農芸化学会  2007/03
• Pseudomonas sp. MIS38株が産生する環状リポペプチドの生成機構の解析  [Not invited]

  鷲尾 健司, Lim Siew-Ping, Roongsawang Niran, 柞木田 なつみ, 森川正章

  農芸化学会  2007/03
• 人に役立つ微生物のちから  [Invited]

  森川 正章

  ヤクルト講演会  2006/11
• 枯草菌のバイオフィルム形成に関する遺伝子と分子  [Invited]

  森川 正章

  日本農芸化学会北海道支部ミニシンポジウム『クオルモン利用の最前線』  2006/11
• Isolation of biofilm-forming bacteria from marine environments  [Not invited]

  M. Morikawa, C. Okada, K. Washio, K. Takano, S. Kanaya

  SGM 159th Meeting  2006/09
• Isolation and characterization of useful bacteria for bioremediation.  [Not invited]

  MORIKAWA, Masaaki

  Tong Hai University-Hokkaido University Joint seminar  2006/08
• Isolation and characterization of bacteria that degrade DEHP.  [Not invited]

  C-M. Chen, K. Washio, K. Takano, S. Kanaya, M. Morikawa

  Tong Hai University-Hokkaido University Joint seminar  2006/08
• バクテリアの個性と社会性 —微生物群集への新たな理解を求めてー「枯草菌のバイオフィルム形成機構」  [Invited]

  森川 正章

  第16回 根圏ネットワーク講演会『バクテリアの個性と社会性』  2006/08
• 疎水環境と微生物  [Invited]

  森川 正章

  バイオウィーク in Sapporo 2006  2006/07
• 特殊環境微生物の最新研究動向と産業利用「疎水環境と微生物」  [Invited]

  森川 正章

  バイオウィーク in Sapporo  2006/07
• 海洋細菌による混合バイオフィルム形成能の評価  [Not invited]

  柞木田なつみ, 岡田千尋, 鷲尾健司, 森川正章

  環境バイオテクノロジー学会  2006/06
• A Novel Biofilm-Forming Rhizobiales Isolated from Seto Inland Sea  [Not invited]

  C. Okada, K. Washio, K. Takano, S. Kanaya, M. Morikawa

  ASM 106th Meeting  2006/05
• Characterization of the C-terminal thioesterase domains in arthrofactin synthetase: a prototype of two internal thioesterases  [Not invited]

  Niran Roongsawang, Kenji Washio, Masaaki Morikawa

  SGM 158th Meeting  2006/04
• 瀬戸内海バイオフィルムから単離された新属細菌の報告  [Not invited]

  岡田千尋, 金谷茂則, 森川正章

  農芸化学会  2006/03
• P12 Structural analysis of a biosurfactant, arthrofactin, produced by Pseudomonas sp. MIS38  [Not invited]

  近藤 泰史, 川上 徹, 森川 正章, 池上 貴久

  産業用酵素国際シンポジウム  2006/02
• Determination of the conformation and configuration of arthrofactin by NMR  [Not invited]

  近藤泰史, 池上貴久, 森川正章

  日本生物物理学会  2005/11
• バイオフィルムの成因と生存メカニズム  [Invited]

  森川 正章

  第12回HACCP・微生物制御に関する講演会  2005/09
• Phylogenetic analysis of condensation domain in nonribosomal peptide synthetases suggests the evolutionary relationship in the peptide donor molecules  [Not invited]

  NIRAN ROONGSAWANG, KAZUFUMI TAKANO, SHIGENORI KANAYA, MASAAKI MORIKAWA

  SGM2005  2005/04
• Identification of epimerase domain in non-ribosomal peptide synthetases (NRPSs) of biosurfactant producing Pseudomonas sp. MIS38  [Not invited]

  Siew Ping Lim, Niran Roongsawang, Kenji Washio, Kazufumi Takano, Shigenori Kanaya, Masaaki Morikawa

  日本農芸化学会2005年大会  2005/03
• Phylogenetic analysis of condensation domain in nonribosomal peptide synthetases suggestes the evolutionary relationship in the peptide donor molecules  [Not invited]

  Niran Roongsawang, Kazufumi Takano, Shigenori Kanaya, Masaaki Morikawa

  日本農芸化学会2005年大会  2005/03
• Studies on Gordonia sihwensis C4 that degrade plasticizer (DEHP)  [Not invited]

  陳 志銘, 森川正章, 高野和文, 金谷茂則

  日本農芸化学会2005年大会  2005/03
• Studies on the biofilm formation by a wild-type Bacillus subtilis  [Not invited]

  森川 正章, 金谷茂則, 今中忠行

  日本生物工学会  2004/11
• Analyses on the alkane degrading genes of Rhodococcus sp. TMP2  [Not invited]

  竹井大亮, 国広波緒, 高野和文, 金谷茂則, 森川正章

  日本生物工学会  2004/11
• Biofilm formation and protease production by Pseudoalteromonas sp. SB-B1  [Not invited]

  森川 正章, 飯島沙織, 岡原良太, 高野和文, 金谷茂則

  日本生物工学会  2004/11
• 好熱性油田細菌と超好熱菌に見る生命の渾沌とした姿  [Invited]

  森川 正章

  日本植物学会北日本支部大会  2004/09
• バイオフィルム  [Invited]

  森川 正章

  サラヤ株式会社セミナー  2003/12
• Complete sequence of arthrofactin biosynthesis operon with unique two carboxy-terminal thioesterase domains.  [Not invited]

  N. Roongsawang, M. Haruki, T. Imanaka, M. Morikawa, S. Kanaya

  ASM 103th  2003/05
• Terrahaemophilus aromaticivorans gen nov. sp. nov., a terrestrial Pasteurellaceae isolated from petroleum sludge.  [Not invited]

  S. Hirano, M. Haruki, T. Imanaka, M. Morikawa, S. Kanaya

  ASM 103th  2003/05
• Oleomonas sagaranensis gen. nov., sp. nov., represents a novel genus in the alpha-Proteobacteria.  [Not invited]

  T. Kanamori, N. Rashid, M. Morikawa, H. Atomi, T. Imanaka

  ASM 103th  2003/05
• Structural analysis of extracellular polymeric substances (EPS) from Bacillus subtilis that forms robust pellicles.  [Not invited]

  M. Morikawa, M. Kagihiro, T. Imanaka, R. Kolter, S. Kanaya

  ASM 103th  2003/05
• 生物の過酷環境への適応戦略ー低温適応を中心として「バイオフィルム形成と環境適応」  [Not invited]

  森川 正章

  大阪大学蛋白質研究所セミナー  2003/03
• Site-specific cleavage of MS2 RNA by a thermostable DNA-linked RNase H.  [Not invited]

  C. Hyongi, T. Nogawa, Y. Tsunaka, M. Haruki, M. Morikawa, S. Kanaya

  RNase H International Conference 2002  2002/09
• Structural and functional analyses of archaeal Type 2 ribonuclease H.  [Not invited]

  A. Muroya, D. Tsuchiya, M. Ishikawa, M. Haruki, M. Morikawa, S. Kanaya, K. Morikawa

  RNase H International Conference 2002  2002/09
• Cleavage of a double-stranded DNA containing a single ribonucleotide at the DNA-RNA junction with prokaryotic RNases HII  [Not invited]

  M. Haruki, Y. Tsunaka, M. Morikawa, S. Kanaya

  RNase H International Conference 2002  2002/09
• 環境に役立つ微生物たち  [Invited]

  森川 正章

  ボストン日本人研究者交流会  2002/01
• The world of extremophilic oil bacteria  [Invited]

  MORIKAWA, Masaaki

  Seminar at Department of Molecular and Cellular Biology, Harvard University  2001/07
• Long-chain-alkane degradation by extremely thermophilic Bacteria.  [Not invited]

  M. Morikawa, T. Kato, M. Haruki, T. Imanaka, S. Kanaya

  ASM 101th  2001/05
• 環境バイオテクノロジー：基礎研究の戦略と事業化への展開  [Invited]

  森川 正章

  日本化学工学会シンポジウム  2001/04
• Biological oil production from CO2.  [Not invited]

  T. Imanaka, M. Morikawa

  Pacifichem 2000  2000/12
• Gene cloning and characterization of an aldehyde dehydrogenase and an esterase from a petroleum degrading strain HD-1.  [Not invited]

  M. Morikawa, S. Mizuguchi, N. Okibe, M. Haruki, T. Imanaka, S. Kanaya

  Pacifichem 2000  2000/12
• An extreme-thermophile which degrades petroleum under high temperature conditions.  [Not invited]

  T. Kato, A. Miyanaga, M. Haruki, T. Imanaka, M. Morikawa, S. Kanaya

  Pacifichem 2000  2000/12
• Thermophilic bacteria isolated from a subterranean petroleum reservoir.  [Not invited]

  M. Morikawa, T. Kato, M. Haruki, T. Imanaka, S. Kanaya

  Gratama Workshop 2000 conference  2000/04
• CO2 fixation and alkane production by an oil-bacterium strain HD-1.  [Invited]

  M. Morikawa, H. Atomi, T. Imanaka

  International Symposium on Autotrophy and Hydrogen Biotechnology  2000/02
• CO2固定細菌を利用した地球環境修復システムの構築  [Invited]

  森川 正章

  近畿バイオインダストリー振興会議第一回技術シーズ公開会  1999/09
• 資源をつくる微生物  [Invited]

  森川 正章

  日本農芸化学会ミニシンポジウム  1998/07
• Isolation of a petroleum degrading/producing bacterium  [Not invited]

  M. Morikawa, S. Jin, K. Amada, M. Haruki, S. Kanaya, T. Imanaka

  International Congress on Extremophiles '98  1998/01
• Petroleum degrading/producing bacterium HD-1 with its unique characters.  [Not invited]

  M. Morikawa, S. Kanaya, T. Imanaka

  Asian Nitrogen-Fixation Symposium under JSPS and Monbusho Programme  1997/12
• A new type of petroleum-degrading/producing bacterium HD-1.  [Not invited]

  M. Morikawa, T. Imanaka

  International Workshop on Ultra-long term Cryogenic Preservation Network of Biological and Environmental Specimens  1997/11
• Production of alkane and alkene from CO2 by a petroleum-degrading bacterium strain HD-1.  [Not invited]

  M. Morikawa, T. Iwasa, S. Yanagida, T. Imanaka

  Fourth International Conference on Carbon Dioxide Utilization  1997/09
• Studies on the structure-function relationship of lipopeptide biosurfactants.  [Not invited]

  M. Morikawa, T. Imanaka

  International Society for Environmental Biotechnology 3rd International Symposium  1996/07
• A new mixotrophic bacterium which can fix CO2 and assimilate aliphatic and aromatic hydrocarbons anaerobically.  [Not invited]

  T. Imanaka, M. Morikawa

  International Society for Environmental Biotechnology 3rd International Symposium  1996/07
• 「極限環境における生体の分子論的応答」有機溶媒：バイオサーファクタントと石油代謝細菌  [Invited]

  森川 正章

  大阪大学蛋白質研究所セミナー  1996/01
• ヒト・リゾチームの構造・機能相関解析  [Invited]

  森川 正章

  生物工学若手研究者の集い  1989/07

Works

• 平成23年度北海道大学大学院地球環境科学研究院公開講座 「生物の環境への適応」

  2011
• オープンキャンパス2011 （北大理学部）

  2011
• プロフェッサービジット2009

  2010
• プロフェッサービジット2010

  2010
• オープンキャンパス2010（北大理学部）

  2010
• ボストン日本人研究者交流会（於，マサチューセッツ工科大学） 「環境に役立つ微生物たち

  2002
• 日本化学工学会シンポジウム 環境バイオテクノロジー：基礎研究の戦略と事業化への展開 展望講演「CO2固定細菌と油田細菌の環境修復技術への可能性」

  2001
• International Symposium on Autotrophy and Hydrogen Biotechnology （於，東京YMCA）"CO2 fixation and alkane production by an oil-bacterium strain HD-1."

  2000
• 近畿バイオインダストリー振興会議第一回技術シーズ公開会 （於，大阪科学技術センター） 「CO2固定細菌を利用した地球環境修復システムの構築

  1999
• 日本農芸化学会ミニシンポジウム（於，大阪府立大学） 「資源をつくる微生物」

  1998
• 大阪大学蛋白質研究所セミナー（於，大阪大学） 「極限環境における生体の分子論的応答」 〜有機溶媒：バイオサーファクタントと石油代謝細菌
• 平成16年度北海道大学大学院地球環境科学研究科公開講座 「地球環境をまもる微生物たち」

MISC

• Game Changing Environmental Purification Technology

  森川正章  Appl. Cell Biol. Japan  34-  33  -54  2021/11
• ウキクサホロビオントが開くグリーンサーキュラーエコノミーへの扉

  森川正章  バイオインダストリー  38-  (7)  2  -9  2021/07  [Invited]
  
• 植物・微生物機能の解析・制御と食糧・バイオマス生産への応用展開

  浅見忠男, 安藤康雄, 森川正章, ほか  日本土壌肥料学雑誌  90-  (1)  82  -87  2019  [Not refereed][Invited]
  

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• ヒトの冬眠は環境バイオテクノロジー？

  森川正章  環境バイオテクノロジー学会ニュースレター  1  -1  2018  [Not refereed][Invited]
  
• グリーンケミストリーの基盤をささえる水生植物の成長を加速する微生物

  森川正章  ケミカルエンジニヤリング  61-  (5)  368  -373  2016/05  [Not refereed][Invited]
  
• 汚水を用いたウキクサバイオマス生産とバイオリファイナリーへの応用

  遠山忠, 森一博, 池道彦, 森川正章  バイオサイエンスとインダストリー  74-  (3)  203  -207  2016/05  [Not refereed][Not invited]
  
• 紅藻マギレソゾLaurencia saitoi由来ブロモペルオキシダーゼの臭素化反応

  正木志良, 金子賢介, 小林大毅, 石川高史, 西川慶祐, 森本善樹, 鷲尾健司, 森川正章, 沖野龍文  日本農芸化学会大会講演要旨集(Web)  2016-  4B032 (WEB ONLY)  2016/03/05  [Not refereed][Not invited]
  
• 紅藻ミツデソゾLaurencia okamurae由来ブロモペルオキシダーゼの性状解析

  石川高史, 金子賢介, 湯暁蓉, 鷲尾健司, 森川正章, 沖野龍文  化学系学協会北海道支部冬季研究発表会講演要旨集(CD-ROM)  2016-  ROMBUNNO.1B17  2016  [Not refereed][Not invited]
  
• 紅藻ソゾ属3種のブロモペルオキシダーゼの活性評価

  金子賢介, 小林大毅, 鷲尾健司, 森川正章, 沖野龍文  日本化学会講演予稿集  95th-  (4)  1409  2015/03/11  [Not refereed][Not invited]
  
• 2P-169 Duckweed growth promotion by Sinorhizobium sp. SP4: transcriptome analysis of the effects of SP4 on duckweed growth

  Toyama Tadashi, Tanaka Yasuhiro, Morikawa Masaaki, Mori Kazuhiro  日本生物工学会大会講演要旨集  67-  217  -217  2015
• 1P-167 Isolation and characterization of novel plant growth promoting bacteria (PGPB) from cormosphere of duckweed

  Iwashita Tomoki, Tanaka Yasuhiro, Tamaki Hideyuki, Makino Ayaka, Toyama Tadashi, Kamagata Youichi, Morikawa Masaaki, Mori Kazuhiro  日本生物工学会大会講演要旨集  67-  130  -130  2015
• 次世代バイオマス：ウキクサの可能性

  森川正章  化学経済  62-  (14)  75  -79  2015  [Not refereed][Invited]
  

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• 廃水を肥料とするバイオマス生産技術の夜明け

  森川正章, 遠山 忠, 田中靖浩, 森 一博, 玉木秀幸, 清 和成, 黒田真史, 三輪京子, 鎌形洋一, 池 道彦  再生と利用  39-  (149)  50  -53  2015  [Not refereed][Invited]
  

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• 3P-149 Screening of Microalgae Growth-Promoting Bacteria

  Quach Angela, Horino Taro, Kuroda Masashi, Toyama Tadashi, Tamaki Hideyuki, Morikawa Masaaki, Ike Michihiko  日本生物工学会大会講演要旨集  67-  308  -308  2015  [Not refereed][Not invited]
  
• 紅藻ウラソゾLaurencia nipponica由来バナジウム依存型ブロモペルオキシダーゼの性状解析

  KANEKO KENSUKE, WASHIO KENJI, UMEZAWA DAIKI, MATSUDA FUYUHIKO, MORIKAWA MASAAKI, OKINO TATSUFUMI  日本水産学会大会講演要旨集  2014-  119  2014/03/27  [Not refereed][Not invited]
  
• P14-13 ウキクサ科植物の葉状体に生息する微生物群集の解析(ポスター発表)

  田中 靖浩, 立野 由佳, 玉木 秀幸, 牧野 彩花, 遠山 忠, 鎌形 洋一, 森川 正章, 森 一博  日本微生物生態学会講演要旨集  2014-  153  -153  2014
• P25-7 水生植物-微生物共生系を構成する新規植物成長促進根圏微生物(ポスター発表)

  牧野 彩花, 玉木 秀幸, 遠山 忠, 田中 靖浩, 森 一博, 池 道彦, 森川 正章, 鎌形 洋一  日本微生物生態学会講演要旨集  2014-  242  -242  2014
• 3P-083 Isolation and characterization of phytobacteria that can highly adhere to duckweed

  Yamakawa Yusuke, Sugawara Masayuki, Ozima Takuya, Miwa Kyoko, Morikawa Masaaki  日本生物工学会大会講演要旨集  66-  215  -215  2014
• 3P-082 Analyses of Microbial Communities Inhabiting Cormosphere and Rhizosphere of Lemnaceae

  Tateno Yuka, Tanaka Yasuhiro, Tamaki Hideyuki, Makino Ayaka, Toyama Tadashi, Kamagata Yoichi, Morikawa Masaaki, Mori Kazuhiro  日本生物工学会大会講演要旨集  66-  215  -215  2014
• 3P-140 The Effects of General Plant Growth-Promoting Compounds on the Growth of duckweed Lemna minor :

  Desi Utami, Masayuki Sugawara, Kyoko Miwa, Masaaki Morikawa  日本生物工学会大会講演要旨集  66-  229  -229  2014
• 2P-137 Groeth promotion of land plant using Plant Growth-Promotion Rizobacteria Acinetobacer calcoaceticus P23

  Kagemoto Keita, Sugawara Masayuki, Ojima Takuya, Miwa Kyoko, Morikawa Masaaki, Toyama Tadashi  日本生物工学会大会講演要旨集  66-  141  -141  2014
• 1P-060 Purification and characterization of novel azo-reductases from Flavobacteriaceae Elizabethkingia sp. RL13212

  Yasukata Kuniki, Murakami Shun, Miwa Kyoko, Morikawa Masaaki  日本生物工学会大会講演要旨集  66-  32  -32  2014
• 新規高度好熱菌Coprothermobacter sp.PM9-2とメタン生成菌の共生に関わる分子機構の解析

  加藤雄大, 漆畑亘, 三輪京子, 森川正章  日本生物工学会大会講演要旨集  66th-  2014
• New Function of the Phytosphere Bacteria from Aquatic Plants

  Masaaki MORIKAWA, Masayuki SUGAWARA, Wakako SUZUKI, Kyoko MIWA  化学と生物  52-  (12)  799  -804  2014  [Not refereed][Invited]
  

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• 紅藻ウラソゾLaurencia nipponica由来バナジウム依存型ブロモペルオキシダーゼの性状解析

  KANEKO KENSUKE, WASHIO KENJI, UMEZAWA DAIKI, MATSUDA FUYUHIKO, MORIKAWA MASAAKI, OKINO TATSUFUMI  マリンバイオテクノロジー学会大会講演要旨集  15th-  68  2013/06/01  [Not refereed][Not invited]
  
• ウキクサ科植物の埋立地浸出水処理への適用性に関する実験的検討

  尾形有香, 石垣智基, 山田正人, 遠山忠, 清和成, 森川正章, 池道彦  全国都市清掃研究・事例発表会講演論文集  35th-  2013
• 1P-163 Exploration and characterization of duckweed rhizobacteria that produce useful compounds

  Mori Shohei, Kurashina Koki, Sugawara Masayuki, Miwa Kyoko, Morikawa Masaaki  日本生物工学会大会講演要旨集  65-  58  -58  2013
• 1P-200 Sinorhizobium sp. SP4 enhances photosynthesis, biomass production and nutrient uptake of duckweed

  Toyama Tadashi, Tanaka Yasuhiro, Mori Kazuhiro, Morikawa Masaaki  日本生物工学会大会講演要旨集  65-  67  -67  2013
• 3P-224 Possibility of duckweed rhizobacterium Acinetobacter sp. P23 for the growth promotion of vegetables

  Suzuki Wakako, Sugawara Masayuki, Miwa Kyoko, Morikawa Masaaki  日本生物工学会大会講演要旨集  65-  244  -244  2013
• 海底油田から単離した高度好熱菌Coprothermobacter sp.PM9-2株の諸特性解析

  加藤雄大, 漆畑亘, 三輪京子, 森川正章, 森川正章  環境バイオテクノロジー学会大会プログラム講演要旨集  2013-  2013
• 3P-149 Sustainable growth promotion of Lemna minor by Acinetobacter calcoaceticus P23 in environmental water

  Hachiya Yoshiyuki, Quach Angela, Tokura Koichiro, Ogata Yuka, Kuroda Masashi, Toyama Tadashi, Morikawa Masaaki, Ike Michihiko  日本生物工学会大会講演要旨集  65-  225  -225  2013  [Not refereed][Not invited]
  
• Unique microbiological characteristics of Pseudoalteromonas spp. that form biofilms in the sea water environment : Their possible contribution to water purification and fish farming.

  Iijima S, Okahara R, Washio K, Morikawa M  Bulletin of the Society of Sea Water Science  66-  186  -190  2012  [Refereed][Not invited]
  
• 4Gp10 Colonization of Acinetobacter calcoaceticus strain P23 on rhizosphere of duckweed, Lemna minor

  HACHIYA Yoshiyuki, QUACH Angela, OGATA Yuka, KURODA Masashi, INOUE Daisuke, MORIKAWA Masaaki, IKE Michihiko  日本生物工学会大会講演要旨集  64-  225  -225  2012  [Not refereed][Not invited]
  
• 1Hp04 Mixed biofilm formation by Marinobacter bacteria and relatives

  Morikawa Masaaki, Eiki Hisashi, Tarakita Natsumi, Washio Kenji  日本生物工学会大会講演要旨集  63-  54  -54  2011
• The role of urease activity on biofilm and urolith formation by Staphylococcus sp T-02 that was isolated from a toilet bowl

  K. Oki, K. Washio, D. Matsui, Y. Hirata, M. Morikawa  JOURNAL OF BIOTECHNOLOGY  150-  S247  -S247  2010/11  [Not refereed][Not invited]
  
• Pseudomonas stutzeriバイオフィルムのナフタレン分解活性と土壌安定性

  嶋田恒平, 片岡剛文, 鷲尾健司, 森川正章  日本農芸化学会大会講演要旨集  2008-  39  2008/03/05  [Not refereed][Not invited]
  
• S-C5 Sustainable bioremediation technology utilizing Biofilms

  MORIKAWA Masaaki, WASHIO Kenji  日本微生物生態学会講演要旨集  (24)  193  -193  2008
• Pseudomonas sutzeriバイオフィルムのナフタレン分解活性と土壌安定性

  嶋田恒平, 片岡剛文, 鷲尾健司, 森川正章  環境バイオテクノロジー学会大会プログラム講演要旨集  31st-  14  2007  [Not refereed][Not invited]
  
• Expression of rice germination-control genes that accompany with the change of DNA modification.

  K Washio, M Morikawa  PLANT AND CELL PHYSIOLOGY  47-  S147  -S147  2006  [Not refereed][Not invited]
  
• 1G17-3 Biofilm formation and protease production by Pseudoalteromonas sp. SB-B1

  IIJIMA Saori, OKAHARA Ryota, TAKANO Kazufumi, KANAYA Shigenori, MORIKAWA Masaaki  日本生物工学会大会講演要旨集  17-  196  -196  2005
• 1A16-3 Analyses on the alkane degrading genes of Rhodococcus sp. TMP2

  TAKEI Daisuke, KUNIHIRO Namio, TAKANO Kazufumi, KANAYA Shigenori, MORIKAWA Masaaki  日本生物工学会大会講演要旨集  17-  56  -56  2005
• 3S4-AM4 Studies on the biofilm formation by a wild-type Bacillus subtilis

  MORIKAWA Masaaki, KANAYA Shigenori, IMANAKA Tadayuki  日本生物工学会大会講演要旨集  17-  47  -47  2005
• Dissection of DNA methylation status in gibberellin-primary response genes that involve the germination of rice seeds.

  K Washio, M Morikawa  PLANT AND CELL PHYSIOLOGY  46-  S130  -S130  2005  [Not refereed][Not invited]
  
• 3P020 Determination of the conformation and configuration of arthrofactin by NMR

  Kondo Y, Morikawa M, Ikegami T  Seibutsu Butsuri  45-  (0)  S208  2005  [Not refereed][Not invited]
  
• 2E11-3 Biofilm bacteria isolated from a fisheries nursery

  OKAHARA Ryota, ISHIABSHI Kanehisa, OKADA Chihiro, TAKANO Kazufumi, MORIKAWA Masaaki, IMANAKA Tadayuki, KANAYA Shigenori  日本生物工学会大会講演要旨集  16-  168  -168  2004
• 2D14-2 Isolation and characterization of microorganisms that degrade plastic elasticizer (DEHP)

  MORIKAWA Masaaki, CHIN Shimei, TAKANO Kazufumi, IMANAKA Tadayuki, KANAYA Shigenori  日本生物工学会大会講演要旨集  16-  156  -156  2004
• リボヌクレアーゼの多様性と酵素機能 Ribonuclease H RNA界とDNA界の間のRibonuclease(Molecular diversities and functions of ribonucleases: RNase H: a ribonuclease between RNA and DNA worlds)

  Tsunaka Yasuo, Chon Hyongi, Takano Kazufumi, Haruki Mitsuru, Morikawa Masaaki, Kanaya Shigenori  生化学  75-  (8)  728  -728  2003/08  [Not refereed][Not invited]
  
• Analysis of a pro-sequence of subtilisin from hyperthermophilic archaeon

  Saito Kenji, Tanaka Shihori, Takano Kazuhumi, Morikawa Masaaki, Kanaya Shigenori  日本生物工学会大会講演要旨集  15-  60  -60  2003
• 2B16-2 Substrate specificity of the internal adenylation domains in arthrofactin synthetase :

  Roongsawang Niran, Takatsuka Atsuko, Takano Kazufumi, Morikawa Masaaki, Kanaya Shigenori  日本生物工学会大会講演要旨集  15-  76  -76  2003
• Biofilm formation and environmental adaptation

  Morikawa Masaaki, Kajihiro Shinji, Haruki Mitsuru, Takano Kazufumi, Imanaka Tadayuki, Kolter Roberto, Kanaya Shigenori  日本生物工学会大会講演要旨集  15-  42  -42  2003
• Analysis of the genes required for the biofilm formation in Bacillus subtilis

  Kawai Yoshitaka, Takano Kazufumi, Morikawa Masaaki, Kanaya Shigenori  日本生物工学会大会講演要旨集  15-  145  -145  2003
• Studies on gentisate 1,2-dioxygenase from polyaromatic hydrocarbon degrading bacterium strain 127W

  Hirano Shinichi, Takano Kazufumi, Imanaka Tadayuki, Morikawa Masaaki, Kanaya Shigenori  日本生物工学会大会講演要旨集  15-  218  -218  2003
• 501 Stability analysis of protein from hyperthermophilic archaeon

  Mukaiyama Atsushi, Chon Hyongi, Takano Kazufumi, Haruki Mitsuru, Morikawa Masaaki, Kanaya Shigenori  日本生物工学会大会講演要旨集  14-  73  -73  2002
• 476 Complete sequence of arthrofactin biosynthesis operon with unique two carboxy-terminal thioesterase domains :

  Roongsawang Niran, Hase Kenichi, Haruki Mitsuru, Morikawa Masaaki, Kanaya Shigenori  日本生物工学会大会講演要旨集  14-  69  -69  2002
• 505 FKBP from Psychrotrophic Bacterium Shewanella sp. SIB1

  Suzuki Yutaka, Haruki Mitsuru, Morikawa Masaaki, Kanaya Shigenori  日本生物工学会大会講演要旨集  14-  74  -74  2002
• 920 Cloning and analysis of PAHs degrading genes from newly isolated bacterium strain 127W

  Hirano Shin-ichi, Haruki Mitsuru, Imanaka Tadayuki, Morikawa Masaaki, Kanaya Shigenori  日本生物工学会大会講演要旨集  14-  169  -169  2002
• 671 Analysis for the function of a C-terminal domain of a family I.3 lipase and its application

  Kuwahara Katsumasa, Kwon Hyun Ju, Haruki Mitsuru, Morikawa Masaaki, Oomori Kenji, Kanaya Shigenori  日本生物工学会大会講演要旨集  14-  112  -112  2002
• 681 Cleavage specificities of prokaryotic type 2 ribonucleases H on DNA-RNA-DNA/DNA chimeric substrates

  Haruki Mitsuru, Morikawa Masaaki, Kanaya Shigenori  日本生物工学会大会講演要旨集  14-  115  -115  2002
• 679 Role of a putative pro-sequence of subtilisin from a hyperthermophilic archaeon

  Tanaka Shihori, Saito Kenji, Haruki Mitsuru, Morikawa Masaaki, Kanaya Shigenori  日本生物工学会大会講演要旨集  14-  114  -114  2002
• 645 Psychrotrophic Type 2 RNase H with unusual enzymatic characteristics

  Chon Hyongi, Ohtani Naoto, Haruki Mituru, Takano Kazufumi, Morikawa Masaaki, Kanaya Shigenori  日本生物工学会大会講演要旨集  14-  106  -106  2002
• 408 Identification and characterization of an oil-degrading bacterium strain HD-1

  Kanamori Takeshi, Rashid Naeem, Morikawa Masaaki, Atomi Haruyuki, Imanaka Tadayuki  日本生物工学会大会講演要旨集  14-  52  -52  2002
• 871 Genetic analysis on biofilm formation by Bacillus subtilis B-1

  Kawai Yoshitaka, Kameyama Takayuki, Morikawa Masaaki, Kanaya Shigenori  日本生物工学会大会講演要旨集  14-  160  -160  2002
• 903 Isolation and characterization of two types of psychrotrophic Rhodococcus strains degrading 2,6,10,14-tetramethylpentadecane (pristane)

  Kunihiro Namio, Haruki Mitsuru, Morikawa Masaaki, Kanaya Shigenori  日本生物工学会大会講演要旨集  14-  165  -165  2002
• 913 Gene expression and characterization of alcohol dehydrogenase from Bacillus thermoleovorans B23

  Nishikawa Hiroshi, Haruki Mitsuru, Morikawa Masaaki, Kanaya Shigenori  日本生物工学会大会講演要旨集  14-  168  -168  2002
• 680 Studies on the catalytic mechanism of Escherichia coli RNase HI in the presence of Mn^<2+>

  Tsunaka Yasuo, Haruki Mitsuru, Oobatake Motohisa, Morikawa Masaaki, Kanaya Shigenori  日本生物工学会大会講演要旨集  14-  114  -114  2002  [Not refereed][Not invited]
  
• 耐熱性ハイブリッドRNase HIによるRNAの配列特異的分解

  全賢基, 津中康央, 春木満, 森川正章, 金谷茂則  生化学  73-  (8)  960  2001/08/25  [Not refereed][Not invited]
  
• Interaction of TIP26 from a hyperthermophilic archaeon with TFB/TBP/DNA ternary complex (vol 5, pg 177, 2001)

  T Matsuda, M Fujikawa, M Haruki, XF Tang, S Ezaki, T Imanaka, M Morikawa, S Kanaya  EXTREMOPHILES  5-  (4)  283  -283  2001/08  [Not refereed][Not invited]
  
• Identification of the histidine and aspartic acid residues essential for enzymatic activity of a family I.3 lipase by site-directed mutagenesis (vol 483, pg 139, 2000)

  HJ Kwon, K Amada, M Haruki, M Morikawa, S Kanaya  FEBS LETTERS  497-  (2-3)  174  -174  2001/05  [Not refereed][Not invited]
  
• Development of an RNA restriction enzyme by using ribonuclease H

  Kanaya Shigenori, Morikawa Masaaki, Haruki Mitsuru, Nogawa Tomoko, Tsunaka Yasuhiro, Chon Hyongi  日本生物工学会大会講演要旨集  13-  12  -12  2001  [Not refereed][Not invited]
  
• 大腸菌リボヌクレアーゼHIの触媒作用における基質のリン酸基の寄与

  春木満, 津中康央, 森川正章, 金谷茂則, 岩井成憲  生化学  72-  (8)  866  2000/08/25  [Not refereed][Not invited]
  
• Gene cloning and characterization of a novel alcohol dehydrogenase from Bacillus thermoleovorans B23.

  Kato Tomohisa, Miyanaga Asuka, Haruki Mitsuru, Morikawa Masaaki, Imanaka Tadayuki, Kanaya Shigenori  日本生物工学会大会講演要旨集  12-  70  -70  2000
• Degradation of aromatic hydrocarbons under microaerobic conditions

  Hirano Shin-ichi, Kitauchi Fumiya, Haruki Mitsuru, Imanaka Tadayuki, Morikawa Masaaki, Kanaya Sigenori  日本生物工学会大会講演要旨集  12-  122  -122  2000
• Exploring of the characteristic metabolism in oil-degrading/producing bacterium strain HD-1 by random sequencing

  Fujikawa Makoto, Haruki Mitsuru, Imanaka Tadayuki, Morikawa Masaaki, Kanaya Sigenori  日本生物工学会大会講演要旨集  12-  132  -132  2000
• Analysis of alkane induced proteins and an aldehyde dehydrogenase from Bacillus thermoleovorans B23.

  Kato Tomohisa, Miyanaga Asuka, Haruki Mitsuru, Morikawa Masaaki, Imanaka Tadayuki, Kanaya Shigenori  日本生物工学会大会講演要旨集  12-  118  -118  2000
• 二価金属イオンを要求しない大腸菌RNase HI変異体の解析

  津中康央, 春木満, 森川正章, 金谷茂則  日本分子生物学会年会プログラム・講演要旨集  22nd-  507  1999/11/22  [Not refereed][Not invited]
  
• Analysis of anaerobic alkane degradation pathway by 2D-PAGE

  Komatsu Ryuta, Matsuda Tomoki, Haruki Mitsuru, Imanaka Tadayuki, Morikawa Masaaki, Kaaya Shigenori  日本生物工学会大会講演要旨集  11-  49  -49  1999
• Studies on calcium dependent lipase from Pseudomonad.

  Amada Kei, Haruki Mitsuru, Morikawa Masaaki, Imanaka Tadayuki, Kanaya Shigenori  日本生物工学会大会講演要旨集  11-  76  -76  1999
• Cloning of Arthrofactin synthetic genes.

  Hase Kenichi, Amada Kei, Nagahisa Keisuke, Haruki Mitsuru, Imanaka Tadayuki, Morikawa Masaaki, Kanaya Shigenori  日本生物工学会大会講演要旨集  11-  140  -140  1999
• Gene cloning and structure analysis of enzymes from a psychrotrophic bacterium Shewanella sp. SIB1

  Miura Keiko, Ohtani Naoto, Sato Akihiro, Kato Tomohisa, Tomoe Yasuyoshi, Haruki Mitsuru, Morikawa Masaaki, Kanaya Shigenori  日本生物工学会大会講演要旨集  11-  141  -141  1999
• Characterization of Bacillus thermoleovorans B23, H41.

  Kato Tomohisa, Morikawa Masaaki, Haruku Mitsuru, Tomoe Yasuyoshi, Imanaka Tadayuki, Kanaya Shigenori  日本生物工学会大会講演要旨集  11-  298  -298  1999
• Degadation of n-alkanes by Bacillus thermoleovorans B23. H41.

  Kato Tomohisa, Miyanaga Asuka, Morikawa Masaaki, Haruki Mitsuru, Tomoe Yasuyoshi, Imanaka Tadayuki, Kanaya Shigenori  日本生物工学会大会講演要旨集  11-  299  -299  1999
• Petroleum production from CO2

  M Morikawa, S Jin, S Kanaya, T Imanaka  NIPPON NOGEIKAGAKU KAISHI-JOURNAL OF THE JAPAN SOCIETY FOR BIOSCIENCE BIOTECHNOLOGY AND AGROCHEMISTRY  72-  (4)  532  -534  1998/04  [Not refereed][Not invited]
  
• Characterization of subtilisin homologue from a hyperthermophilic archaeon

  Inoue Y., Morikawa M., Haruki M., Takagi M., Imanaka T., Kanaya S.  日本生物工学会大会講演要旨集  10-  280  -280  1998
• 1156 Characterization of petroleum degrading bacteria from oil contaminated sea and land in Vietnam :

  Huy Nguyen Quang, Jin Sha, Amada Kei, Morikawa Masaaki, Haruki Mitsuru, Imanaka Tadayuki, Kanaya Shigenori  日本生物工学会大会講演要旨集  10-  295  -295  1998
• Degradation of Crude Oil under Anoxic Conditions by a Newly Isolated Bacterium

  Jin Sha, Morikawa Masaaki, Haruki Mitsuru, Imanaka Tadayuki, Kanaya Shigenori  日本生物工学会大会講演要旨集  10-  294  -294  1998
• Isolation and characterization of novel bacteria from oil fields.

  Kato Tomohisa, Funayama Tetsu, Morikawa Masaaki, Haruki Mitsuru, Tomoe Yasuyoshi, Imanaka Tadayuki, Kanaya Shigenori  日本生物工学会大会講演要旨集  10-  294  -294  1998
• Characterization of a newly isolated thermophilic bacterium from an oil field under the bottom sea.

  Funayama Tetsu, Morikawa Masaaki, Haruki Mitsuru, Imanaka Tadayuki, Kanaya Shigenori  日本生物工学会大会講演要旨集  10-  295  -295  1998
• Gene cloning and characterization of aldehyde dehydrogenase from petroleum dissimilating/assimilating bacterium HD-1

  Okibe Naoko, Morikawa Masaaki, Amada Kei, Haruki Mitsuru, Imanaka Tadayuki, Kanaya Shigenori  日本生物工学会大会講演要旨集  10-  123  -123  1998  [Not refereed][Not invited]
  
• Gene cloning and characterization of lipase from biosurfactant producing strain Arthrobacter sp. MIS38.

  Amada Kei, Morikawa Masaaki, Imanaka Tadayuki, Kanaya Shigenori  日本生物工学会大会講演要旨集  9-  12  -12  1997
• Development of a culture system for the CO_2 fixation and alkane production by microorganisms.

  Jin Sha, Morikawa Masaaki, Funayama Tetsu, Haruki Mitsuru, Imanaka Tadayuki, Kanaya Shigenori  日本生物工学会大会講演要旨集  9-  246  -246  1997
• Preparation of transcriptional initiation complex by using hyperthermophilic archaeal TATA binding protein as affinity ligand.

  Matsuda Tomoki, Morikawa Masaaki, Haruki Mitsuru, Imanaka Tadayuki, Kanaya Shigenoli  日本生物工学会大会講演要旨集  9-  158  -158  1997
• Cloning of aldehyde dehydrogenase gene from photosynthetic bacterium Rhodospirillum rubrum

  Okibe Naoko, Morikawa Masaaki, Amada Kei, Haruki Mitsuru, Imanaka Tadayuki, Kanaya Shigenori  日本生物工学会大会講演要旨集  9-  12  -12  1997  [Not refereed][Not invited]
  
• 超好熱始原菌Pyrococcus sp. KOD1 由来 Glycerol Kinase のクローニングと解析

  古賀 雄一, 森川 正章, 春木 満, 今中 忠行, 金谷 茂則  日本分子生物学会年会プログラム・講演要旨集  19-  136  -136  1996/08
• Thermus thermophilus RNase H C末端欠損変異体の熱安定性解析

  平野 展孝, 春木 満, 森川 正章, 大畠 玄久, 金谷 茂則  日本分子生物学会年会プログラム・講演要旨集  19-  716  -716  1996/08/01
• 微生物による石油分解と石油生成 (特集/バイオテクノロジ-最近の話題)

  森川 正章, 今中 忠行  化学工業  47-  (5)  365  -371  1996/05
• Isolation of an esterase producing bacterium and cloning of the gene.

  Jinno Kazuhito, Morikawa Masaaki, Matsumoto Shusaku, Imanaka Tadayuki  日本生物工学会大会講演要旨集  8-  186  -186  1996
• 454 RecA homologue from a hyperthermophilic archaeon maintains the major domain only :

  Rashid Naeem, Morikawa Masaaki, Kanaya Shigenori, Imanaka Tadayuki  日本生物工学会大会講演要旨集  8-  123  -123  1996
• Surface-chemical characterization of arthrofactin.

  Morikawa Masaaki, Hirata Yoshihiko, Horiuchi Teruo, Ohdera Motoyasu, Imanaka Tadayuki  日本生物工学会大会講演要旨集  8-  290  -290  1996
• Isolation of dibenzothiophene degradation bacteria from petroleum.

  Kitauchi Fumiya, Morikawa Masaaki, Imanaka Tadayuki  日本生物工学会大会講演要旨集  8-  253  -253  1996
• Characterization and improvemont of petroleum producing bacterium strain HD-1.

  Morikawa Masaaki, Ikeda Masaru, Nagahisa Keisuke, Haruki Mitsuru, Kanaya Shigenori, Imanaka Tadayuki  日本生物工学会大会講演要旨集  8-  252  -252  1996
• 細菌によるCO_2固定と石油生産(2) : 環境科学

  森川 正章, 岩佐 毅, 清水 一朗, 永久 圭介, 柳田 祥三, 今中 忠行  日本農藝化學會誌  69-  377  -377  1995/07/05
• 超好熱始原菌KOD1株由来DNAポリメラーゼの特性 : 酵素

  北林 雅夫, 荒川 琢, 井上 浩明, 川上 文清, 川村 良久, 高木 昌宏, 森川 正章, 今中 忠行  日本農藝化學會誌  69-  246  -246  1995/07/05
• Studies on the structure-activity relationship of biosurfactants.

  Hirata Yoshihiko, Morikawa Masaaki, Imanaka Tadayuki  日本生物工学会大会講演要旨集  7-  264  -264  1995
• Genome analysis of 16S rRNA flanking region of Pyrococcus sp. KOD1.

  Morikawa Masaaki, Fujiwara Shinsuke, Imanaka Tadayuki  日本生物工学会大会講演要旨集  7-  215  -215  1995
• Studies on the membrane domain of signal sensor protein ComP.

  Pongpobpibool Sutee, Morikawa Masaaki, Imanaka Tadayuki  日本生物工学会大会講演要旨集  6-  221  -221  1994
• Studies on the structure-activity relationship of biosurfactant.

  Hirata Yoshihiko, Morikawa Masaaki, Imanaka Tadayuki  日本生物工学会大会講演要旨集  6-  214  -214  1994
• 656 Cloning and Analysis of 16 S-rRNA gene and Transcriptional Factor TF IID gene from Hyperthermophilic Archaeum strain KOD 1 :

  Rashid Naeem, Morikawa Masaaki, Imanaka Tadayuki  日本生物工学会大会講演要旨集  6-  236  -236  1994
• Characterization of amylase pullulanase from thermophilic and alkaliphilic Bacillus sp.

  Lee Sang Pil, Morikawa Masaaki, Takagi Masahiro, Imanaka Tadayuki  日本生物工学会大会講演要旨集  6-  87  -87  1994
• Cloning and analsis of the DNA polymerase gene from a new hyperthermophilc archaeon KOD1

  Kakihara Hirofumi, Takagi Masahiro, Morikawa Masaaki, Imanaka Tadayuki  日本生物工学会大会講演要旨集  5-  168  -168  1993
• Degradation of saturated aliphatic hydrocarbon by P__-. anaerooleophila HD-1

  Kanemoto Mitsuhide, Morikawa Masaaki, Imanaka Tadayuki  日本生物工学会大会講演要旨集  5-  144  -144  1993
• Isolation and Application of Thermophilic Bacteria

  Imanaka Tadayuki, Takagi Masahiro, Morikawa Masaaki  日本生物工学会大会講演要旨集  5-  10  -10  1993
• Purification and characterization of a thermostable thiol protease from Pyrococcus sp. KOD1

  伊澤 啓文, 八木 正貴, 森川 正章, 今中 忠行, 帆秋 利洋  日本生物工学会大会講演要旨集  5-  224  -224  1993  [Not refereed][Not invited]
  
• Cloning and expression of anti-porphyrin monoclonal antibody gene in Escherichia coil.

  Ueda Makoto, Hamuro Takuya, Morikawa Masaaki, Harada Akira, Kamati Mikiharu, Imanaka Tadayuki  日本生物工学会大会講演要旨集  4-  30  -30  1992
• Cloning and sequencing of the gene for surfactin production.

  Morikawa Masaaki, Suzuki Masanori, Goto Shinya, Ito Mikito, Imanaka Tadayuki  日本生物工学会大会講演要旨集  4-  34  -34  1992
• Isolation of a new biosurfactant producer and structure analysis of its product.

  Daido Hiromi, Morikawa Masaaki, Imanaka Tadayuki  日本生物工学会大会講演要旨集  4-  217  -217  1992
• Examination of culture condition for P__-. anaerooleophila HD-1.

  Morikawa Masaaki, Sato Yoshiaki, Imanaka Tdayuki  日本生物工学会大会講演要旨集  4-  165  -165  1992
• Cloning of PPlase gene from Bacillus stearothermophilus SIC1

  Kim Dongju, Morikawa Masaaki, Imanaka Tadayuki  日本生物工学会大会講演要旨集  4-  125  -125  1992
• Isolation and characterization of hyper-thermophilic archaebacteria from hot spring.

  Izawa Yoshifumi, Kakihara Hirofumi, Morikawa Masaaki, Imanaka Tadayuki, Hoaki Toshihiro  日本生物工学会大会講演要旨集  4-  207  -207  1992  [Not refereed][Not invited]
  
• Electron microscopic observation of Pseudomonas anaerooilphilus HD-1

  Morikawa Masaaki, Imanaka Tadayuki  日本醗酵工学会大会講演要旨集  3-  114  -114  1991
• Isolation of a facultative anaerobe which can grow anaerobicaly on n-tetradecane as a sole carbon source.

  Morikawa Masaaki, Imanaka Tadayuki  日本醗酵工学会大会講演要旨集  2-  166  -166  1990

Industrial Property Rights

• 特願2014-148493:植物成長強化剤及びそれを用いた植物栽培方法  2014年/07/22

  鈴木 和歌子, 菅原 雅之, 三輪 京子, 森川 正章, 玉木 秀幸, 牧野 彩花, 鎌形 洋一
• 特願WO2017002929A1:植物の生長を促進する方法、植物生長促進物質を製造する方法及びこれらに利用されるタンパク質  2015年/06/30

  森川正章, ジョグ ラフル, 菅原雅之, 三輪京子
• 特許第4955039号:土壌汚染浄化方法    2012/03/23

  長谷川武, 平本弘, 山本英樹, 岡崎修, 森川正章
• 特開2011-030487:キノン類の製造方法  2011/02/17

  森川正章, 大胡康, 鈴木良雄  

  特願2009-179141
• 特開2009-247279:新規水草根圏微生物  2009/10/29

  森川正章, 鷲尾健司, 山賀文子
• 特許第3507890号:Ｓｈｅｗａｎｅｌｌａｓｐ．ＳＩＢ１株由来の新規なアルカリフォスファターゼ    2004/01/09

  金谷 茂則, 森川 正章, 春木 満
• 特許第3120114号:アルカン類を分解する新規微生物、アルカン類を分解する方法  

  森川 正章, 加藤 智久
• 特開2000-354496:核酸の増幅方法およびその試薬  2000/12/26

  北林 雅夫, 荒川 琢, 井上 浩明, 川上 文清, 川村 良久, 今中 忠行, 高木 昌宏, 森川 正章
• 特開2000-23687:熱安定性ＤＮＡポリメラ―ゼ  2000/01/25

  北林 雅夫, 荒川 琢, 井上 浩明, 川上 文清, 川村 良久, 今中 忠行, 高木 昌宏, 森川 正章
• 特開平11-056349:蔗糖脂肪酸エステル合成酵素を産生する微生物  1999/03/02

  今中 忠行, 森川 正章, 西畑 隆男, 神野 和人
• 特開平11-056364:薯糖脂肪酸エステラーゼ  1999/03/02

  今中忠行, 森川正章, 西畑隆男, 神野和人
• 特開平11-32773:耐熱性グリセロールキナーゼ及びそれをコードするＤＮＡ  1999/02/09

  金谷茂則, 森川正章
• 特開平10-306098:環状リポペプチド化合物の化学合成方法および環状リポペプチド化合物  1998/11/17

  今中 忠行, 平田 善彦, 森川 正章
• 特許第3120114号:新規微生物  

  森川正章, 金 沙
• 特開平8-322597:核酸の増幅方法およびその試薬  1996/12/10

  北林 雅夫, 荒川 琢, 井上 浩明, 川上 文清, 川村 良久, 今中 忠行, 高木 昌宏, 森川 正章
• 特開平7-298879:超好熱始原菌由来のＤＮＡポリメラーゼ遺伝子およびその用途  1995/11/14

  今中 忠行, 高木 昌宏, 森川 正章, 柿原 博文
• 特願平5-352901:シュードモナス属細菌を用いた炭化水素の生産方法及び新規シュードモナス属細菌  1993年/12/29

  今中 忠行, 森川 正章, 桜井 尚二
• 特開平5-276933:新規微生物  1993/10/26

  森川 正章
• 特開平5-211876:バイオサーファクタントの濃縮方法  1993/08/24

  桜井 尚二, 今中 忠行, 森川 正章
• 特開平5-211892:バイオサーファクタント産生菌のスクリーニング方法及びバイオサーファクタントの活性測定方法  1993/08/24

  桜井 尚二, 真鍋 洋平, 今中 忠行, 森川 正章
• 特開平1-120286:改変ヒト・リゾチーム、それをコードする遺伝子及び改変ヒト・リゾチームの製造方法  1989/05/12

  村木三智郎, 地神芳文, 田中秀明, 森川正章
• 特願2015-130895:植物の生長を促進する方法、植物生長促進物質を製造する方法及びこれらに利用されるタンパク質  

  森川 正章
• 特開昭62-263192:卵黄レシチンの製造方法  

  森川正章, 徳永恵美子, 堺宗雄

Awards & Honors

• 2022/11 日本水処理生物学会 Paper award
   Isolation and Characterization of Novel Plant Growth-Promoting Bacteria from the Fronds of Duckweed 

  受賞者: Iwashita T;Tanaka Y;Tamaki H;Nakai R;Yoneda Y;Makino A;Toyama T;Kamagata Y;Morikawa M;Mori K
• 2022/10 Kasetsart University, Thailand Honorary Doctorate of Philosophy (Bioscience)
  
• 2019/11 日本水処理生物学会 Paper award
   Biomass production and nutrient removal through cultivation of Euglena gracilis in domestic wastewater 

  受賞者: Masashi Kuroda;Taro Horino;Daisuke Inoue;Masaaki Morikawa;Michihiko Ike
• 2017 ISCDRA First Place “2017 Knowing to Growing Duckweed Application Award”
   Evaluation of environmental bacterial communities as a factor affecting the growth of duckweed Lemna minor 

  受賞者: H. Ishizawa;M. Kuroda;M. Morikawa;M. Ike
• 2011 日本農芸化学会 第8回農芸化学研究企画賞
  
• 2007 天野エンザイム 第8回 酵素応用シンポジウム研究奨励賞
  
• 2003 バイオインダストリー協会 奨励賞
   発酵と代謝
• 1999 日本生物工学会 第7回生物工学論文賞
   

  受賞者: 森川 正章;岩佐 毅;柳田 祥三;今中 忠行;阪大院;工;京大院
• 1994 日本生物工学会 第2回生物工学論文賞
   

  受賞者: 森川 正章;今中 忠行
• 1993 日本生物工学会 第1回生物工学論文賞
   

  受賞者: 森川 正章;伊東 幹人;今中 忠行

Research Grants & Projects

• Elucidation of holobiont co-evolution mechanism by analyzing aquatic plant-surface symbiotic bacteria interactions

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2021/07 -2024/03 

  Author : 森川 正章
• Development of the duckweed holobiont resource values towards Thailand BCG economy

  Japan Science and Technology Agency/Japan Internationas Cooperation Agency：Science and Technology Research Partnership for Sustainable Development

  Date (from‐to) : 2020/08 

  Author : Masaaki Morikawa
• Effective Aquatic Biomass Production Utilizing Mutualistic Microorganisms

  Japan Science and Technology Agency：Advanced Low Carbon Technology and Research Development

  Date (from‐to) : 2016/09 -2020/03 

  Author : MORIKAWA, Masaaki
• 新奇な水生植物ー微生物相互作用の発見と理解

  JSPS:科学研究補助金：挑戦的萌芽研究

  Date (from‐to) : 2016/04 -2018/03 

  Author : 森川 正章
• Application of Rhizosphere Microorganisms Symbiosis to Highly Ordered Plant BIoprocesses

  Japan Science and Technology Agency：Advanced Low Carbon Technology Research and Development Program

  Date (from‐to) : 2011/10 -2016/08 

  Author : Masaaki Morikawa
• 高度好熱菌性油田細菌のアルカン代謝および生態に関する研究

  JSPS：科学研究費基盤B一般

  Date (from‐to) : 2010 -2012 

  Author : 森川 正章
• バイオフィルム工学による微生物のデザイン化

  NEDO：受託研究「微生物機能を活用した環境調和型製造基盤技術開発」

  Date (from‐to) : 2007 -2011 

  Author : 森川 正章
• バイオフィルム形成分子機構を切り口とした微生物未知機能の解明

  JSPS：科学研究費基盤B一般

  Date (from‐to) : 2007 -2009 

  Author : 森川 正章
• バイオフィルムを利用した環境修復技術の開発

  （財）発酵研究所：発酵研究所研究助成

  Date (from‐to) : 2006 -2008 

  Author : 森川 正章
• 寒冷条件に対応した次世代汚染土壌修復技術の開発

  METI：受託研究 （中小企業ベンチャー挑戦支援事業)

  Date (from‐to) : 2008 

  Author : 森川 正章
• 非リボソーム型ペプチド合成酵素の解析と機能改変

  （株）天野エンザイム：酵素応用シンポジウム奨励賞

  Date (from‐to) : 2007 

  Author : 森川 正章
• 脂溶性機能性食品成分の菌体外分泌システム開発

  （財）食生活研究会：食生活研究会研究助成

  Date (from‐to) : 2007 

  Author : 森川 正章
• バイオフィルム工学を利用した生物環境修復技術の開発

  J-Power：共同研究 （J-Power ５０周年記念先端技術共同研究)

  Date (from‐to) : 2005 -2006 

  Author : 森川 正章
• Arthrofactin 合成反応の分子メカニズム

  JSPS：科学研究費基盤C一般

  Date (from‐to) : 2005 -2006 

  Author : 森川 正章
• 病原微生物データ分析実験作業

  （財）食品産業センター：受託研究 食品製造工程管理情報高度化促進事業

  Date (from‐to) : 2006 

  Author : 森川 正章
• 新規リボソーム非依存型ペプチド合成酵素の構造・機能相関に関する研究

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2004 -2005 

  Author : 森川 正章, ROONGSAWANG Niran

   

  非リボソーム型ペプチド合成酵素は生理活性のある非常に多様な複合/環状ペプチド類を合成することができる。合成最終段階の環状化反応と産物の遊離は合成酵素のC末端に存在するチオエステラーゼドメイン(TE)の働きにより進行する。Pseudomonas属細菌MIS38が有するarthrofactin合成酵素は3つのサブユニット(ArfA/B/C)から構成される。興味深いことにArfCのC末端には2つのTEが存在する。いずれのTE(TE1,TE2)も活性発現に必須のSr/Asp/Hisを有している。本研究では、これらのTEが合成反応にどのように係わっているのかを解明することを目的とした。 当該遺伝子部分を大腸菌用ベクターを用いてクローニングした後、PCR法を用いて部位特異的突然変異遺伝子を構築した。さらにこの変異遺伝子を相同性組み換え法によりMIS38染色体に戻した。こうして、S91A,S91T,S383A,S383/D410A変異株を作製した。塩基配列解析を行い、それぞれの位置以外に突然変異が導入されていないことを確認した。こうして得られた変異株のarthrofactin生産性を調べた。その結果、S91A,S91T変異株は生産性を完全に失っていた。一方、S383AおよびS383A/D410A変異株は野生株に比べて5±1%の活性を保持していた。またこれら変異株が生産するarthrofactinは野生株のものと同一の構造であることを確認した。以上の結果から、S91を含むTE1が環状化反応と産物の遊離に必須であり、TE2は本質的に重要ではないことが判明した。現在,この仮説を証明するためにTE2を完全に決失した変異株を作成中である。
• 汚染土壌微生物生態を利用した環境修復技術の開発

  住友財団：

  Date (from‐to) : 2005 

  Author : 森川 正章
• 革新的リボソーム非依存型複合ペプチド合成酵素に関する研究

  武田科学振興財団：武田科学振興財団奨励金一般研究

  Date (from‐to) : 2005 

  Author : 森川 正章
• カラー空間符号モアレマッチング法によるゲノム情報における規則性の抽出

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2003 -2003 

  Author : 谷田 純, 森川 正章

   

  空間符号モアレマッチング技術を基本としたゲノム解析手法の開発を通して,その有効性,特牲,問題点を明らかにするとともに,ゲノム情報処理分野における情報フォトニクス技術応用の可能性を探ることを目的とする.本年度に得られた知見は以下の通りである. 1.昨年度考案したカラー空間符号モアレマッチング技術を実装したプログラム(MTerm)により,各種細菌類を対象とした全ゲノム比較を行い,有効性,特性,問題点を評価した.ほとんどの比較において一様なモアレ縞が観測され,モアレ縞が観測されないほうがむしろ稀であることを確認した.また,出現頻度符号化を用いた場合,区切り枠の取り方により一致線が分裂する現象を見いだし,その対策として,MTermに区切り枠の調整機構を付加した. 2.ゲノム配列内に存在する逆位の検出性に対する検討を行った.配列塩基種の相補塩基への変換,塩基の配置方向の反転などの機能をMTerm付加した.大腸菌O157EDL933株と同sakai株に適用し,配列多重度10000においても逆位部位の検出が行えることを確認した. 3.配列一致部位の自動検出をめざして,出力モアレ縞画像に対する特徴抽出処理の検討を行った.移動平均法の多重適用,閾値処理による二値化,膨張・収縮処理によるノイズ成分除去の一連の処理により,良好な特徴抽出結果が得られることを確認した.さらに,処理画像に対する情報圧縮により,1/500程度の圧縮率が得られることを確認した.
• Development of heat-labile and psychrophilic enzymes for clean and efficient recombinant DNA experiment

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2002 -2003 

  Author : HARUKI Mitsuru, MORIKAWA Masaaki

   

  We tried to develop heat-labile alkaline phosphatase, BglII, and RNase A to inactivate these enzymes by heat in recombinant DNA experiment, and to isolate psychrophilic DNA ligase gene for efficient DNA ligation at low temperatures. An alkaline phosphatase (APase) from psychrotrophic bacterium Shewanella sp. SIBI was overproduced in E. coli, purified, and characterized. The enzyme exhibited the highest activity at 40℃. This optimum temperature is shifted downward by 20℃, as compared to that of E. coli APase. SIBI APase was almost fully inactivated upon incubation at 80℃. for 5min, whereas E. coli APase retained 60% of the activity upon incubation at 80℃ for 1h. The k_ value of SIBI APase was higher than that of E. coli APase by 6.2 times at 40℃ and 9.6 times at 20℃. A part of DNA ligase gene was isolated from Shewanella sp. SIB1. A pair of PCR primers was designed based on the DNA sequence of the DNA ligase gene of Shewanella oneidensis MR-1, which is closely related to Shewanella sp. SIB1. A 600-bp DNA fragment was amplified by PCR using MR-1 genomic DNA as a template and the designed primers. The DNA fragment encoded an amino acid sequence with 77% identity to MR-1 DNA ligase. Isolation of the entire gene is now under progress. To select heat-labile mutant of Bgl II and RNase A, we tried to display these enzymes on M13 phage by inserting the genes of these enzymes with a 6 × His-tag at their N-termini to the 5'-terminus of the coat protein pIII gene. Phagemid particles were produced from E. coli strains harboring these hybrid genes, and were analyzed for the binding to a Ni-NTA plate. However, no specific binding was observed, possibly because phage display of these enzymes was unsuccessful.
• 空間符号モアレマッチング法によるマルチスケールゲノム情報解析

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2002 -2002 

  Author : 谷田 純, 森川 正章

   

  空間符号化法を用いた配列比較技術に対して,符号化法の改良や専用視覚化端末の開発を通し,全ゲノム配列比較への拡張手法に関する種々の検討を行った.また,これらの手法を各種大腸菌の全ゲノムやヒト染色体などの実配列に適用し,その有効性を評価した.その結果,以下に示す成果を得た. 1.単一ディスプレイ上でマッチング処理を実行するためのソフトウェアを開発し,これまでに考案した種々の符号化法を実装した.さらに,符号化画像の更新や選択配列部位の切り出しなどの様々な機能を付加させることにより,専用視覚端末の利便性を向上させた. 2.長大な2配列間のマッチング結果を同時に提示するため,対象配列を一定の長さを有する部分配列に分割し,部分配列内の各塩基要素を符号パターンに反映させる出現頻度符号化法を考案した.この符号化法を用いることで,数10万塩基長に及ぶ2配列間のマッチング結果を同時に表示できることを確認した. 3.出現頻度符号化法により得られる出力結果から,配列間の部分一致性や欠失・挿入だけでなく,配列内の塩基組成分布に関する様々な情報を抽出できることを新たに見出した.さらに,出現頻度符号の配色を工夫することで,DNA配列解析において重要であるGC含有量を出力結果から直感的に把握できることを実験的に確認した. 4.出現頻度符号化法、およびその部分配列長の可変機能を専用端末上に実装し,微視レベルから巨視レベルまでのマルチスケールな配列比較を統合して扱える環境を構築した.この符号化法を用いて,大腸菌K-12株と病原性大腸菌O-157:H7株を比較し,全ゲノム配列から部分一致性を抽出することに成功した.
• 高温溶媒系で生きる細胞の戦略

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2001 -2001 

  Author : 森川 正章
• 空間符号モアレマッチング法による鳥瞰的ゲノム情報解析

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2001 -2001 

  Author : 谷田 純, 森川 正章

   

  配列情報の空間符号化を利用した配列間マッチング技術に対して,符号化規則の変更や出力モアレ縞の解釈によって得られるゲノム配列間の情報抽出法を検討した.これらの手法を,公開ゲノム配列データベースより取得したDNA及び,アミノ酸配列に適用し,評価を行った.その結果,以下に示す成果を得た. 1.モアレ情報端末の利用性向上のため,液晶ディスプレイ上に2配列の符号化画像を表示する方式を検討し,所望のマッチング結果を得ることに成功した.これより,コンピュータ制御による簡易視覚端末が実現できることを確認した. 2.アミノ酸配列間の類縁性を含めた比較情報を抽出するため,疎水性パラメータを空間符号に導入する手法を考案し,弱相関配列の検出を試みた.既存のホモロジー検索ツール,モチーフ抽出ツールとの比較から,アミノ酸配列比較技術としての有効性を示した. 3.ドットプロット法との比較により,提案手法が有する特長を明らかにした.モアレ現象の特性により適度な情報抽出が行われ,DNA配列に対してもフィルタリング処理なしで有用な情報が抽出できる点,表示に必要なデータ数を減少でき,高速かつ簡便にデータ更新が行える点,検査範囲が等しければ,より高い視認性で出力が得られる点などである. 4.高密度符号化法を開発し,10万要素の同時比較の可能性を示すことができた.いくつかの特定の縦列反復配列部位において観察される特徴的な2次元モアレパターンを明らかにした.また,処理手順を整理し,自己相関相互相関,反転相関の有用性を示した.これらの成果より,2配列間の類似性を鳥瞰的に視覚化する手法の体系化を行い,提案手法のもつ多様な情報抽出の可能性を提示した.
• 空間符号モアレ法に基づく光学的ゲノム情報処理技術の開発

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2000 -2000 

  Author : 谷田 純, 森川 正章

   

  光演算技術は,大容量情報に対する並列処理性と,人間の優れたパターン認識能力を誘起する情報可視性をあわせ持ち,それらの有効利用はゲノム情報処理の効率化に寄与しうる.本研究では,その具体的手法として,空間符号モアレ法による2配列間マッチング演算を対象とし,特性,および,有効性を評価した. 空間符号モアレ法では,符号パターンや実現方法により,出力情報の加工が可能である.相補配列の検出能力をもつ対称型符号パターンを用いて,ステムループ構造の抽出が行えることを確認した.次に,濃度反転符号と非反転符号の組合せによる一致信号の先鋭化技術を考案し,従来方式では不可能であった1塩基置換によるミスマッチの検出を実現した.また,3色の符号パターンにより,3配列間マッチング処理を実現した. 一方,検討手法の簡便な利用をめざして,マッチング情報端末を試作した.2台の液晶ディスプレイ画面を半透鏡で合成する方式と,1台の液晶ディスプレイ画面に印刷済透明シートを張り合わせる方式とを実装した.後者の方式は,データ操作の自由度では劣るものの,視認性の点で極めて優れている. 光演算技術に基づく情報システムとして,自由空間光インターコネクションによる配列マッチング専用プロセッサの基本構成を検討した.空間符号を時分割展開した時間符号が処理スループットの点で優れていることを見い出し,予備実験により原理確認を行った. 空間符号モアレ法は,ホモロジー検索の基本演算であるドット・プロット法と等価な信号を出力する.しかし,モアレ縞は,各要素のマッチング結果が継ぎ目なく表されるアナログ画像情報である.その結果,人間のパターン認識能力をより強く誘起しうる可能性をもつ.符号化については,視覚特性を考慮する必要がある.一つの展開方向として,動態視の利用によりマッチング識別能力を向上させる方式が考えられる.
• Studies on degradation mechanism of petroleum under anoxic condition.

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 1996 -1997 

  Author : MORIKAWA Masaaki

   

  Bacterial strain HD-1, isolated from an oil field, is able to degrade both aliphatic and aromatic hydrocarbons anaerobically. In this project, bacterial degradation mechanisms of petroleum under anoxic condition was studied Utilizing tetradecane (alkane) as a substrate, two kinds of alkene were accumulated as degradation intermediates in HD-1 cells. Finally a main component of the alkenes was identified as 1-dodecene based on analyzes data from FT-IR,1H-NMR,and GC/MS.This result shows that alkane is dehydrogenated by the strain HD-1 under anoxic condition. Although one example was reported in 1992 from a research group in Germany about alkane degradation under anoxic condition, this is the first report on the structural analysis of a degradation intermediate. Moreover, strain HD-1 were found to be able to grow on CO2 as a sole carbon source and produce alkane in the cells. Its production yield was 0.1% in dry cell weight. The metabolic pathway from CO2 to alkane needs to be elucidated in a future. Recently, a new bacterial strain TK-122 was isolated oil-reservoir tank. Its oil degradation ability is superior to HD-1 under both oxic and anoxic conditions. About 70% of alkanes (from octane to hexadecane) in petroleum, was degraded in 3 days under anoxic condition. Molibdenate (MoO4^<2->) was used as an electron acceptor in the anaerobic degradation of alkane. This result indicates a finding of a new petroleum degradation pathway. According to the analysis on 16S rRNA nucleotide sequence of the strain TK-122, Escherichia or Citrobacter was most probable genus. However, physiological characterization of the strain matched with none of these genera. It is indicated that the strain TK-122 belongs to a new genus.
• CO_2固定と石油生成に関する微生物学的アプローチ

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 1995 -1995 

  Author : 森川 正章, 今中 忠行

   

  【1】アルカン/アルケン生合成経路の解明 まず、生物学的にCO_2からアルカン/アルケンを合成する経路のなかで最も研究が遅れており、実際反応律速になっている可能性が高いと思われる脂肪酸からアルカン/アルケンへの変換反応について検討した。緑藻類などを用いた研究結果からは脂肪酸から直接アルカン/アルケンを合成しているのではなく、脂肪酸からアルデヒドになった後アルカン/アルケンに変換される可能性が示唆されている。そこでHD-1株が最も多く蓄積していたヘキサデカン(C16)の前駆物質であると予想されるパルミチン酸あるいはヘキサデカナ-ルを使ってアルカン/アルケンの生成が実際に起こるかを調べた。^<14>C-パルミチン酸は市販のものを利用した。^<14>C-ヘキサデカナ-ルは入手不可能であったため^<14>C-パルミチン酸から化学合成した。アルカン/アルケンの生成反応は以下のようにして行った。基質である^<14>C-パルミチン酸あるいは^<14>C-ヘキサデカナ-ルを含むリン酸緩衝液(pH7.0)/1% Triton X-100をナスフラスコ内でArガス通気により脱酸素処理する。同様に脱酸素処理した細胞抽出液を嫌気性ボックス内で添加後密栓する。これを遮光した湯溶中で37℃24時間保温した。反応産物を含む疎水性画分をクロロホルム抽出し、気質のみで保温したコントロールと共にシリカゲル60TLC(ヘキサンおよびヘキサン,ジエチルエーテル,ギ酸)で展開し、脂肪酸あるいはアルデヒド画分(Rf=0.5-0.7)とアルカン/アルケン画分(Rf=0.9以上)を厳密に分けて回収した。液体シンチレーションカウンターによりそれぞれの放射活性を測定した。この結果から、微量ではあるが細胞抽出液を加えた場合にのみアルカン/アルケンの生成が確認できた。さらに脂肪酸にくらべてアルデヒドの方がアルカン/アルケンの生成率が良いことか、細菌においても脂肪酸はアルデヒドを経由してアルカン/アルケンに変換されることが強く示唆された。現在細胞抽出液をカラムクロマトグラフィーなどにより分画して、本酵素活性(アルデヒドデカルボニラーゼ)の精製を目指している。 【2】アルカン/アルケン合成能力の向上 本プロジェクトが実際に地球環境レベルで貢献できるにはHD-1株の生育速度を高めると共にアルカン/アルケンの蓄積量を飛躍的に増大する必要がある。そこで、人為的突然変異処理を繰り返してこの目的に合った変異株を効率良く選別する方法を検討した。突然変異処理法は細菌において最も強力で汎用的なニトロソグアニジン処理を採用した。処理条件は生存率10%〜50%を設定(100mg/1,37℃,30分間)した。アルカン/アルケンの合成能力の高くなった株はプレート上では元株と容易には区別できない。そこで比重の違いを利用した。すなわちアルカン/アルケンを含む疎水性成分の細胞内含量が高いものは他にくらべて軽くなっていると予想した。比重の違いで生体物質を分画するのに最も簡便な方法のひとつが密度勾配遠心分離法である。そこで細胞を分画するための無菌的密度勾配遠心分離の最適条件検討を策定した。遠心分離後、上層から注意深く溶液を10区画(No.1〜No.10)に分けて回収した。これをプレートに広げて単一細胞を単離して最も比重の軽い細胞(No.5)、中程度の細胞(No.7)、最も重い細胞(No.10)を選んでそれぞれの大量培養後、疎水性成分含量を比較した。取得したNo.5は変異剤未処理の約3倍の疎水性物質を蓄積していた。現在ガスクロマトグラフィーにより疎水性成分(アルカン/アルケン含量など)を分析中である。以上の結果から、突然変異処理(ニトロソグアニジンおよび紫外線照射)と密度勾配遠心分離による選別により、アルカン/アルケンの高生産性突然変異株の取得が可能であることが確認できた。今後、この操作を繰り返すことによってHD-1株のアルカン/アルケン生成能力を可能な限り増強させる予定である。
• Characterization of Anaerobic Petroleum Degrading Bacterium strain HD-1.

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 1993 -1995 

  Author : IMANAKA Tadayuki, MORIKAWA Masaaki

   

  We isolated a new type of Gram-negative mixotrophic bacterium, Pseudomonas sp. HD-1, which is a facultative anaerobe. Cells were grown on CO_2 as a sole carbon source in the presence of H_2. Anaerobic growth of the strain was enhanced by the addition of aliphatic as well as aromatic hydrocarbons. When cells were grown on n-alkane anaerobically, many oil vacuoes formed in the cells and about 40-50% of the dry cell weight was lipid and oil. The total increase in the hydrophilic components of the cells indicated the anaerobic assimilation of aliphatic hydrocarbon. Electron microscopic observation showed that the cell surface was highly wavy and that the membrane had a multi-layr structure irrespective of the presence of oil. The strain was demonstrated to degrade tetradecane (C_<14>) under anaerobic condition and one of the major metabolic intermediates was identified as 1-dodecene (C_<12>). This result demonstrates that alkanes are biodegradable without molecular oxygen via a pathway different from beta-oxidation. The strain was found to grow on CO_2 in the presence of tetradecane and absence of H_2 gas and light. These facts strongly indicate that the bacterium is able to utilize alkane as an energy source anaerobically.
• CO_2固定と石油生成に関する微生物学的アプローチ

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 1994 -1994 

  Author : 森川 正章, 今中 忠行

   

  原材料をCO_2ガス、最終産物を石油成分(特にパラフィン系炭化水素)とする一連のプロセス(炭素物質循環系)の構築を前提として油田土壌から分離されたHP-1株代謝経路の解明ならびに菌株を改良することが本研究の目的である。本年度はHP-1株がCO_2から炭化水素を生成できることの確認とその代謝経路の推定、および遺伝子組み換え法により本菌株を改良するための基盤を確立することを目的とした。 先ず、CO_2を単一炭素源とする培地0.5%(NH_4_2SO_4,0.1%KH_2PO_4,0.05%MgCl_2・6H_2O/水道水(pH7.0)にCO_2を含む混合嫌気ガス(CO_2:H_2:N_2=5:5:90)を通気して37℃2週間培養した。増殖した菌体を遠心分離により回収・洗浄したのち凍結乾燥した。クロロホルム抽出によって調製した疎水性画分についてGC分析を行った結果、パラフィンに相当する位置にピークが検出されたため、さらにGC-MSによる質量数の決定ならびに構造の推定を行った。親イオンピークならびにフラグメントイオンピークの特徴から脂肪酸(パルミチン酸、ステアリン酸)以外に質量数278と226のパラフィン(それぞれ、エイコサジエンならびにヘキサデカン)が確認できた。以上の結果、HP-1株はCO_2とH_2から石油主成分であるパラフィンを生産することを確認した。 次に、本菌のCO_2固定能力ならびにパラフィン合成能力を強化するために遺伝子組み換え基盤技術の確立をめざした。汎用プラスミドベクター14種類についてエレクトロポレーション法によりHD-1株の形質転換を試みたところ、グラム陰性細菌用ベクターRSF1010(Sm^r)によりパルス条件10kV/cm 1 mses2回により2.3×10^3/μg DNA の頻度で形質転換可能であることを見いだした。本研究成果によりCO_2固定経路とパラフィン合成経路における律速段階を触媒する酵素遺伝子群を遺伝子工学的に強化できる見通しがついた言える。それぞれの代謝経路についてその機構を解析することが今後の課題である。
• Petroleum degrading and producing bacteria (under low temperature, low oxygen conditions) Bioremediation technology (Encocrine disruptant degrading bacteria) Biosynthetic pathways of biosurfactant (Non-ribosomal peptide synthetase) Biofilm formation (M・・・

  Petroleum degrading and producing bacteria (under low temperature, low oxygen conditions) Bioremediation technology (Encocrine disruptant degrading bacteria) Biosynthetic pathways of biosurfactant (Non-ribosomal peptide synthetase) Biofilm formation (Marine bacteria, Bacillus, Hyperthermophiles)

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• 2023/06 - Today   Japan Society for Environmental Biotechnology   President
• 2005/09 - Today   Higher Education Commision, Pakistan   Foreign Experts
• 2015/06 -2023/05   Japanese Society for Environmental Biotechnology   Vice-president