研究者データベース

研究者情報

マスター

アカウント(マスター)

  • 氏名

    大井 俊彦(オオイ トシヒコ), オオイ トシヒコ

所属(マスター)

  • 工学研究院 応用化学部門 生物工学分野

所属(マスター)

  • 工学研究院 応用化学部門 生物工学分野

独自項目

syllabus

  • 2020, 応用生化学特論, Advanced Applied Biochemistry, 修士課程, 総合化学院, 遺伝情報,タンパク質構造,分子論的理解,生合成機構,動物細胞,二次代謝産物,バイオポリマー,環境浄化
  • 2020, 生命分子化学特論, Modern Trends in Biomolecular Chemistry, 修士課程, 総合化学院, 遺伝情報,タンパク質構造,分子論的理解,生合成機構,動物細胞,二次代謝産物,バイオポリマー,環境浄化
  • 2020, 大学院共通授業科目(一般科目):自然科学・応用科学, Inter-Graduate School Classes(General Subject):Natural and Applied Sciences, 修士課程, 大学院共通科目, 遺伝情報,タンパク質構造,分子論的理解,生合成機構,動物細胞,二次代謝産物,バイオポリマー,環境浄化
  • 2020, 生命分子化学特論, Modern Trends in Biomolecular Chemistry, 修士課程, 工学院, 遺伝情報,タンパク質構造,分子論的理解,生合成機構,動物細胞,二次代謝産物,バイオポリマー,環境浄化
  • 2020, 生命分子化学特論, Modern Trends in Biomolecular Chemistry, 博士後期課程, 工学院, 遺伝情報,タンパク質構造,分子論的理解,生合成機構,動物細胞,二次代謝産物,バイオポリマー,環境浄化
  • 2020, 一般教育演習(フレッシュマンセミナー), Freshman Seminar, 学士課程, 全学教育, バイオテクノロジー、遺伝子工学、遺伝子組換え技術、環境バイオテクノロジー、化学プロセスと生物プロセス、酵素・微生物の医療・産業応用
  • 2020, 生化学Ⅱ, Biochemistry II, 学士課程, 工学部, 遺伝情報,遺伝子発現,複製,転写,翻訳,遺伝子工学,クローニング,塩基配列分析,遺伝子増幅,タンパク質工学
  • 2020, 物質変換工学, Applied Chemistry and Biochemistry, 学士課程, 工学部, 有機合成,有機材料,化学プロセス,反応器設計,生体材料,高分子材料,分子機能,無機材料,複合材料,電子材料,光機能材料
  • 2020, 応用化学学生実験Ⅲ, Applied Chemistry Laboratory Ⅲ, 学士課程, 工学部, 微生物の培養,微生物の観察,遺伝子工学,形質転換,大量発現,タンパク質精製

researchmap

プロフィール情報

学位

  • 農学博士(大阪府立大学)

プロフィール情報

  • 大井, オオイ
  • 俊彦, トシヒコ
  • ID各種

    201401073034846087

対象リソース

業績リスト

研究キーワード

  • セルラーゼ   キメラ酵素   糖質加水分解酵素   キシラナーゼ   Active Site   バイオマス   構造と機能   Aspergillus   Cellulase   Bacillus pumilus   Aspergillus aculeatus   Fungi   Aspergillug aculeatus   微生物工場   バイオプラスチック   バイオリファイナリー   アゾレダクターゼ   enzyme   Aeromonas sp.   azo dye   Trichoderma sp.   アゾ染料   微生物分解   Bacillus sp.   コンゴーレッド   オレンジII   gene   アゾ色素   脱色酵素   azoreductase   

研究分野

  • ライフサイエンス / 応用微生物学
  • 環境・農学 / 環境農学
  • ものづくり技術(機械・電気電子・化学工学) / バイオ機能応用、バイオプロセス工学
  • 環境・農学 / 環境材料、リサイクル技術
  • 環境・農学 / 環境負荷低減技術、保全修復技術

経歴

  • 2014年 北海道大学 工学(系)研究科(研究院) 准教授

論文

  • Shuzo Arai, Sayaka Sakakibara, Robin Mareschal, Toshihiko Ooi, Manfred Zinn, Ken'ichiro Matsumoto
    Frontiers in bioengineering and biotechnology 8 612991 - 612991 2020年 
    Glycolate (GL)-containing polyhydroxyalkanoate (PHA) was synthesized in Escherichia coli expressing the engineered chimeric PHA synthase PhaC AR and coenzyme A transferase. The cells produced poly[GL-co-3-hydroxybutyrate (3HB)] with the supplementation of GL and 3HB, thus demonstrating that PhaC AR is the first known class I PHA synthase that is capable of incorporating GL units. The triad sequence analysis using 1H nuclear magnetic resonance indicated that the obtained polymer was composed of two distinct regions, a P(GL-ran-3HB) random segment and P(3HB) homopolymer segment. The random segment was estimated to contain a 71 mol% GL molar ratio, which was much greater than the value (15 mol%) previously achieved by using PhaC1 P s STQK. Differential scanning calorimetry analysis of the polymer films supported the presence of random copolymer and homopolymer phases. The solvent fractionation of the polymer indicated the presence of a covalent linkage between these segments. Therefore, it was concluded that PhaC AR synthesized a novel random-homo block copolymer, P(GL-ran-3HB)-b-P(3HB).
  • Isolation of poly[D-lactate (LA)-co-3-hydroxybutyrate)]-degrading bacteria from soil and characterization of D-LA homo-oligomer degradation by the isolated strain.
    Hori C, Sugiyama T, Watanabe K, Sun J, Kamada Y, Ooi T, Isono T, Satoh T, Sato S, Taguchi S, Matsumoto K
    Polymer Degradation and Stability 2020年 [査読有り][通常論文]
  • Maho Sudo, Chiaki Hori, Toshihiko Ooi, Shoji Mizuno, Takeharu Tsuge, Ken'ichiro Matsumoto
    Journal of bioscience and bioengineering 129 3 302 - 306 2019年10月18日 [査読有り][通常論文]
     
    The engineered chimeric polyhydroxyalkanoate (PHA) synthase PhaCAR is composed of N-terminal portion of Aeromonas caviae PHA synthase and C-terminal portion of Ralstonia eutropha (Cupriavidus necator) PHA synthase. PhaCAR has a unique and useful capacity to synthesize the block PHA copolymer poly(2-hydroxybutyrate-block-3-hydroxybutyrate) [P(2HB-b-3HB)] in engineered Escherichia coli from exogenous 2HB and 3HB. In the present study, we initially attempted to incorporate the amino acid-derived 2-hydroxyalkanoate (2HA) units using PhaCAR and the 2HA-CoA-supplying enzymes lactate dehydrogenase (LdhA) and CoA transferase (HadA). Cells harboring the genes for PhaCAR, LdhA, and HadA, as well as for the 3HB-CoA-supplying enzymes β-ketothiolase and acetoacetyl-CoA reductase, were cultivated with supplementation of four hydrophobic amino acids, i.e., leucine, valine (Val), isoleucine (Ile), and phenylalanine, in the medium. No hydrophobic amino acid-derived monomers were incorporated into the polymer, which was most likely because of the strict substrate specificity of PhaCAR; however, P(2HB-co-3HB) was unexpectedly produced with Val supplementation. The copolymer was likely P(2HB-b-3HB) based on proton nuclear magnetic resonance analysis. Based on the endogenous pathways in E. coli, 2HB units are likely derived from threonine (Thr) through deamination and dihydroxylation. In fact, dual supplementation with Thr and Val showed synergy on the 2HB fraction of the polymer. Val supplementation promoted the 2HB synthesis likely by inhibiting the metabolism of 2-ketobutyrate into Ile and/or activating Thr dehydratase. In conclusion, the LdhA/HadA/PhaCAR pathway served as the system for the synthesis of P(2HB-b-3HB) from biomass-derived carbon sources.
  • Matsumoto K, Saito J, Yokoo T, Hori C, Nagata A, Kudoh Y, Ooi T, Taguchi S
    Journal of bioscience and bioengineering 128 3 302 - 306 2019年09月 [査読有り][通常論文]
  • Hori C, Yamazaki T, Ribordy G, Takisawa K, Matsumoto K, Ooi T, Zinn M, Taguchi S
    Journal of bioscience and bioengineering 2018年12月 [査読有り][通常論文]
  • Matsumoto K, Iijima M, Hori C, Utsunomia C, Ooi T, Taguchi S
    Biomacromolecules 19 7 2889 - 2895 2018年07月 [査読有り][通常論文]
  • Hori C, Oishi K, Matsumoto K, Taguchi S, Ooi T
    Journal of bioscience and bioengineering 125 6 632 - 636 2018年06月 [査読有り][通常論文]
  • Kadoya R, Matsumoto K, Takisawa K, Ooi T, Taguchi S
    Journal of bioscience and bioengineering 125 4 365 - 370 2018年04月 [査読有り][通常論文]
  • Matsumoto K, Hori C, Fujii R, Takaya M, Ooba T, Ooi T, Isono T, Satoh T, Taguchi S
    Biomacromolecules 19 2 662 - 671 2018年02月 [査読有り][通常論文]
  • Ken'ichiro Matsumoto, Kurato Yamazaki, Shun Kawakami, Daichi Miyoshi, Toshihiko Ooi, Shigeki Hashimoto, Seiichi Taguchi
    SCIENTIFIC REPORTS 7 12136 - 12136 2017年09月 [査読有り][通常論文]
     
    Identifying the target molecules of antimicrobial agents is essential for assessing their mode of action. Here, we propose Acquired Resistance induced by Gene Overexpression (ARGO) as a novel in vivo approach for exploring target proteins of antimicrobial agents. The principle of the method is based on the fact that overexpression of the expected target protein leads to reduced sensitivity to the antimicrobial agent. We applied this approach to identify target proteins of the antimicrobial peptide apidaecin, which is specifically effective against Gram-negative bacteria. To this end, a set of overexpression Escherichia coli clones was tested, and peptide chain release factor 1, which directs the termination of translation, was found as a candidate, suggesting that apidaecin inhibits the termination step of translation. This finding was confirmed in vivo and in vitro by evaluating the inhibitory activity of apidaecin towards lacZ reporter gene expression, which is tightly dependent on its stop codon. The results of this study demonstrate that apidaecin exerts its antimicrobial effects partly by inhibiting release factors.
  • Daisuke Ishii, Kenji Takisawa, Ken'ichiro Matsumoto, Toshihiko Ooi, Takaaki Hikima, Masaki Takata, Seiichi Taguchi, Tadahisa Iwata
    POLYMER 122 169 - 173 2017年07月 [査読無し][通常論文]
     
    Poly[(R)-lactate-co-(R)-3-hydroxybutyratel [P(LA-co-3HB)] is a biobased polyester with flexible properties efficiently synthesized by engineered Escherichia coli. Here, we aimed at optimizing the monomeric composition of the copolymer in terms of its flexibility, and elucidating structural features contributing to their mechanical properties. The IA fraction was successfully regulated in the range of 6-66 mol% by combination of metabolic and enzyme engineering approaches. The copolymers with higher LA fraction showed decreasing melting point from 160 to 125 degrees C, but increasing glass transition temperature from 7 to 27 degrees C. Crystallinity of the as-cast film, that was mainly attributed to the crystallization of 3HB unit, also decreased with the increasing LA fraction. Owing to the combined effect of these parameters, the highest elongation to break (approximately 400%), which is comparable to that of polyethylene, was obtained for the copolymers with middle range LA fraction (33 mol%). The high flexibility was maintained in most of the copolymers. Notably, P(21mol%LA-co-3HB) retained its high elongation at break (about 300%) even after storage under ambient condition for 5 months. These results demonstrate that introduction of LA units into the polymer chain effectively and stably inhibited the crystallization of 3HB units. (C) 2017 Elsevier Ltd. All rights reserved.
  • Ryosuke Kadoya, Yu Kodama, Ken'ichiro Matsumoto, Toshihiko Ooi, Seiichi Taguchi
    JOURNAL OF BIOSCIENCE AND BIOENGINEERING 123 5 535 - 539 2017年05月 [査読有り][通常論文]
     
    Engineered Escherichia coli is a useful platform for production of lactate (LA)-based polyester poly[LA-co-3-hydroxybutyrate (3HB)] from renewable sugars. Here we screened all non-lethal transcription factor deletions of E. coli for efficient production of the polymer. This approach aimed at drawing out the latent potential of the host for efficient polymer production via indirect positive effects. Among 252 mutants from Keio Collection tested, eight mutants (Delta pdhR, Delta cspG, Delta yneJ, Delta chbR, Delta yiaU, Delta creB, Delta ygfI and Delta nanK) accumulated greater amount of polymer (6.2-10.1 g/L) compared to the parent strain E. coli BW25113 (5.1 g/L). The mutants increased polymer production per cell (1.1-1.5-fold) without significant change in cell density. The yield of the polymer from glucose was also higher for the selected mutants (0.34-0.38 g/g) than the parent strain (0.27 g/g). Therefore, the deletions of transcription factors should channel the carbon flux towards polymer production. It should be noted that the screening employed in this study identified beneficial mutants without analyzing causal relationship between the mutation and the enhanced polymer production. This approach, therefore, should be applicable to broad range of fermentation productions. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.
  • Kenji Takisawa, Toshihiko Ooi, Ken'ichiro Matsumoto, Ryosuke Kadoya, Seiichi Taguchi
    PROCESS BIOCHEMISTRY 54 102 - 105 2017年03月 [査読有り][通常論文]
     
    Woody extract-derived hemicellulosic hydrolysate, which was obtained from dissolving pulp manufacturing, was utilized as feedstock for the production of poly(lactate-co-3-hydroxybutyrate) P(LA-co-3HB)] in engineered Escherichia coil. The hydrolysate was composed of mainly xylose and galactose, and contained impurities mainly acetate, which was found to inhibit the polymer synthesis rather than the cell growth. Thus, acetate and other impurities were removed through active charcoal and ion-exchange columns. Using the purified hydrolysate, P(LA-co-3HB) was successfully produced (cell dry weight 8.6 g/L, polymer concentration 5.4 g/L, LA fraction 5.5 mol%, polymer content 62.4%), the amount of which was comparable to that obtained using reagent grade xylose and galactose. Therefore, the hydrolysate from woody extract is considered as an abundant, inexpensive and efficient feedstock applicable to consolidated process for P(LA-co-3HB) production, when the removal of acetic acid was satisfactorily accomplished. (C)2016 Elsevier Ltd. All rights reserved.
  • Jian Sun, Camila Utsunomia, Shohei Sasaki, Ken’ichiro Matsumoto, Toshihiko Yamada, Toshihiko Ooi, Seiichi Taguchi
    Bioscience, Biotechnology, and Biochemistry 80 4 818 - 820 2016年04月 [査読有り][通常論文]
  • Jian Sun, Ken'ichiro Matsumoto, Yuta Tabata, Ryosuke Kadoya, Toshihiko Ooi, Hideki Abe, Seiichi Taguchi
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 99 22 9555 - 9563 2015年11月 [査読有り][通常論文]
     
    Polyhydroxyalkanoate depolymerase derived from Variovorax sp. C34 (PhaZ(Vs)) was identified as the first enzyme that is capable of degrading isotactic P[67 mol% (R)-lactate(LA)-co-(R)-3-hydroxybutyrate(3HB)] [P(d-LA-co-d-3HB)]. This study aimed at analyzing the monomer sequence specificity of PhaZ(Vs) for hydrolyzing P(LA-co-3HB) in comparison with a P(3HB) depolymerase from Alcaligenes faecalis T1 (PhaZ(Af)) that did not degrade the same copolymer. Degradation of P(LA-co-3HB) by action of PhaZ(Vs) generated dimers, 3HB-3HB, 3HB-LA, LA-3HB, and LA-LA, and the monomers, suggesting that PhaZ(Vs) cleaved the linkages between LA and 3HB units and between LA units. To provide a direct evidence for the hydrolysis of these sequences, the synthetic methyl trimers, 3HB-3HB-3HB, LA-LA-3HB, LA-3HB-LA, and 3HB-LA-LA, were treated with the PhaZs. Unexpectedly, not only PhaZ(Vs) but also PhaZ(Af) hydrolyzed all of these substrates, namely PhaZ(Af) also cleaved LA-LA linkage. Considering the fact that both PhaZs did not degrade P[(R)-LA] (PDLA) homopolymer, the cleavage capability of LA-LA linkage by PhaZs was supposed to depend on the length of the LA-clustering region in the polymer chain. To test this hypothesis, PDLA oligomers (6 to 40 mer) were subjected to the PhaZ assay, revealing that there was an inverse relationship between molecular weight of the substrates and their hydrolysis efficiency. Moreover, PhaZ(Vs) exhibited the degrading activity toward significantly longer PDLA oligomers compared to PhaZ(Af). Therefore, the cleaving capability of PhaZs used here toward the d-LA-based polymers containing the LA-clustering region was strongly associated with the substrate length, rather than the monomer sequence specificity of the enzyme.
  • Seiichi Taguchi, Toshihiko Ooi, Kouhei Mizuno, Hiromi Matsusaki
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 99 22 9349 - 9360 2015年11月 [査読有り][通常論文]
     
    The choice of an appropriate microbial host cell and suitable production conditions is crucial for the downstream processing of pharmaceutical- and food-grade products. Although Escherichia coli serves as a highly valuable leading platform for the production of value-added products, like most Gram-negative bacteria, this bacterium contains a potent immunostimulatory lipopolysaccharide (LPS), referred to as an endotoxin. In contrast, Gram-positive bacteria, notably Bacillus, lactic acid bacteria (LAB), Corynebacterium, and yeasts have been extensively used as generally recognized as safe (GRAS) endotoxin-free platforms for the production of a variety of products. This review summarizes the currently available knowledge on the utilization of these representative Gram-positive bacteria for the production of eco- and bio-friendly products, particularly natural polyesters, polyhydroxyalkanoates, bacteriocins, and membrane proteins. The successful case studies presented here serve to inspire the use of these microorganisms as a main-player or by-player depending on their individual properties for the industrial production of these desirable targets.
  • Ryosuke Kadoya, Ken'ichiro Matsumoto, Toshihiko Ooi, Seiichi Taguchi
    PLOS ONE 10 6 e0125163  2015年06月 [査読有り][通常論文]
     
    Bacterial polyester polyhydroxyalkanoates (PHAs) have been produced in engineered Escherichia coli, which turned into an efficient and versatile platform by applying metabolic and enzyme engineering approaches. The present study aimed at drawing out the latent potential of this organism using genome-wide mutagenesis. To meet this goal, a transposon-based mutagenesis was carried out on E. coli, which was transformed to produce poly (lactate-co-3-hydroxybutyrate) from glucose. A high-throughput screening of polymer-accumulating cells on Nile red-containing plates isolated one mutant that produced 1.8-fold higher quantity of polymer without severe disadvantages in the cell growth and monomer composition of the polymer. The transposon was inserted into the locus within the gene encoding MtgA that takes part, as a non-lethal component, in the formation of the peptidoglycan backbone. Accordingly, the mtgA-deleted strain E. coli JW3175, which was a derivate of superior PHA-producing strain BW25113, was examined for polymer production, and exhibited an enhanced accumulation of the polymer (7.0 g/l) compared to the control (5.2 g/l). Interestingly, an enlargement in cell width associated with polymer accumulation was observed in this strain, resulting in a 1.6-fold greater polymer accumulation per cell compared to the control. This result suggests that the increase in volumetric capacity for accumulating intracellular material contributed to the enhanced polymer production. The mtgA deletion should be combined with conventional engineering approaches, and thus, is a promising strategy for improved production of intracellularly accumulated biopolymers.
  • Enhanced cellular content and lactate fraction of the P(lactate-co-3-hydroxybutyrate) produced in recombinant Escherichia coli by the deletion of sigma factor RpoN
    R. Kadoya, Y. Kodama, K. Matsumoto, T. Ooi, S. Taguchi
    J. Biosci. Bioeng. 119 4 427 - 429 2015年04月 [査読有り][通常論文]
     
    組換え大腸菌を用いた乳酸ポリマー生産を効率化するため,大腸菌の遺伝子のマスタースイッチであるσ因子を欠失させた変異体を用いて,ポリマー生産性を評価した。その結果,窒素枯渇条件で活性化されるRpoNを欠失させた変異株が,乳酸ポリマーの生産性およびポリマー中の乳酸分率が上昇することがわかった。特に,窒素枯渇条件になると考えられる培養後期にポリマー生産速度が落ちにくい事が,全体のポリマー量の上昇につながった。
  • Jian Sun, Ken'ichiro Matsumoto, John Masani Nduko, Toshihiko Ooi, Seiichi Taguchi
    POLYMER DEGRADATION AND STABILITY 110 44 - 49 2014年12月 [査読無し][通常論文]
     
    Poly[(R)-lactate-co-(R)-3-hydroxybutyratel [P(LA-co-3HB)] is a biobased polyester with semitransparent and flexible properties produced in engineered bacteria carrying an LA-polymerizing enzyme. In this study, we attempted to isolate the P(enriched LA-co-3HB)-degrading bacteria from soil samples in order to identify enzymes with the capacity to degrade this new type of polymer. Among approximately 500 samples, the Gram-negative bacterium 04, which exhibited potent P(enriched LA-co-3HB)-degrading activity, was isolated based on the decrease in the turbidity of culture medium supplemented with emulsified P(67 mol% LA-co-3HB). Based on its 16S rDNA sequence, this isolated bacterium was identified as a member of Variovorax sp.. Next, we attempted to isolate and purify the depolymerase that contributes to the polymer degradation from the culture supernatant of strain C34. The purified enzyme had a molecular mass of 42 kDa and exhibited degradation activity towards the P(67 mol% LA-co-3HB) as well as 3HB homopolymer [P(3HB)], but not LA homopolymers (PDLA and PLLA). On the other hand, a well-characterized depolymerase of P(3HB) derived from Alcaligenes faecalis T1 did not degrade P(67 mol% LA-co-3HB), PDLA or PLLA. This result suggests that the newly isolated depolymerase differs from the P(3HB) depolymerase from A. faecalis. (C) 2014 Elsevier Ltd. All rights reserved.
  • Ken'ichiro Matsumoto, Kota Tobitani, Shunsuke Aoki, Yuyang Song, Toshihiko Ooi, Seiichi Taguchi
    AMB EXPRESS 4 1 83  2014年11月 [査読有り][通常論文]
     
    The biosynthesis of poly(lactic acid) (PLA)-like polymers, composed of >99 mol% lactate and a trace amount of 3-hydroxybutyrate, in engineered Corynebacterium glutamicum consists of two steps; the generation of the monomer substrate lactyl-coenzyme A (CoA) and the polyhydroxyalkanoate (PHA) synthase-catalyzed polymerization of lactyl-CoA. In order to increase polymer productivity, we explored the rate-limiting step in PLA-like polymer synthesis based on quantitative metabolite analysis using liquid chromatography mass spectroscopy (LC-MS). A significant pool of lactyl-CoA was found during polymer synthesis. This result suggested that the rate-limitation occurred at the polymerization step. Accordingly, the expression level of PHA synthase was increased by means of codon-optimization of the corresponding gene that consequently led to an increase in polymer content by 4.4-fold compared to the control. Notably, the codon-optimization did not significantly affect the concentration of lactyl-CoA, suggesting that the polymerization reaction was still the rate-limiting step upon the overexpression of PHA synthase. Another important finding was that the generation of lactyl-CoA was concomitant with a decrease in the acetyl-CoA level, indicating that acetyl-CoA served as a CoA donor for lactyl-CoA synthesis. These results show that obtaining information on the metabolite concentrations is highly useful for improving PLA-like polymer production. This strategy should be applicable to a wide range of PHA-producing systems.
  • John Masani Nduko, Ken'ichiro Matsumoto, Toshihiko Ooi, Seiichi Taguchi
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 98 6 2453 - 2460 2014年03月 [査読有り][通常論文]
     
    Poly(lactate-co-3-hydroxybutyrate) (P(LA-co-3HB)) was previously produced from xylose in engineered Escherichia coli. The aim of this study was to increase the polymer productivity and LA fraction in P(LA-co-3HB) using two metabolic engineering approaches: (1) deletions of competing pathways to lactate production and (2) overexpression of a galactitol transporter (GatC), which contributes to the ATP-independent xylose uptake. Engineered E. coli mutants (Delta pflA, Delta pta, Delta ackA, Delta poxB, Delta dld, and a dual mutant; Delta pflA + Delta dld) and their parent strain, BW25113, were grown on 20 g l(-1) xylose for P(LA-co-3HB) production. The single deletions of Delta pflA, Delta pta, and Delta dld increased the LA fraction (58-66 mol%) compared to BW25113 (56 mol%). In particular, the Delta pflA + Delta dld strain produced P(LA-co-3HB) containing 73 mol% LA. Furthermore, GatC overexpression increased both polymer yields and LA fractions in Delta pflA, Delta pta, and Delta dld mutants, and BW25113. The Delta pflA + gatC strain achieved a productivity of 8.3 g l(-1), which was 72 % of the theoretical maximum yield. Thus, to eliminate limitation of the carbon source, higher concentration of xylose was fed. As a result, BW25113 harboring gatC grown on 40 g l(-1) xylose reached the highest P(LA-co-3HB) productivity of 14.4 g l(-1). On the other hand, the Delta pflA + Delta dld strain grown on 30 g l(-1) xylose synthesized 6.4 g l(-1) P(LA-co-3HB) while maintaining the highest LA fraction (73 mol%). The results indicated the usefulness of GatC for enhanced production of P(LA-co-3HB) from xylose, and the gene deletions to upregulate the LA fraction in P(LA-co-3HB). The polymers obtained had weight-averaged molecular weights in the range of 34,000-114,000.
  • Jian Yu, Daiki Ogata, ZuoQi Gai, Seiichi Taguchi, Isao Tanaka, Toshihiko Ooi, Min Yao
    ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY 70 Pt 2 553 - 564 2014年02月 [査読有り][通常論文]
     
    Azo dyes are major synthetic dyestuffs with one or more azo bonds and are widely used for various industrial purposes. The biodegradation of residual azo dyes via azoreductase-catalyzed cleavage is very efficient as the initial step of wastewater treatment. The structures of the complexes of azoreductases with various substrates are therefore indispensable to understand their substrate specificity and catalytic mechanism. In this study, the crystal structures of AzrA and of AzrC complexed with Cibacron Blue (CB) and the azo dyes Acid Red 88 (AR88) and Orange I (OI) were determined. As an inhibitor/analogue of NAD(P) H, CB was located on top of flavin mononucleotide (FMN), suggesting a similar binding manner as NAD(P) H for direct hydride transfer to FMN. The structures of the AzrC-AR88 and AzrC-OI complexes showed two manners of binding for substrates possessing a hydroxy group at the ortho or the para position of the azo bond, respectively, while AR88 and OI were estimated to have a similar binding affinity to AzrC from ITC experiments. Although the two substrates were bound in different orientations, the hydroxy groups were located in similar positions, resulting in an arrangement of electrophilic C atoms binding with a proton/electron-donor distance of similar to 3.5 angstrom to N5 of FMN. Catalytic mechanisms for different substrates are proposed based on the crystal structures and on site-directed mutagenesis analysis.
  • Ken'ichiro Matsumoto, Takehiro Okei, Inori Honma, Toshihiko Ooi, Hirobumi Aoki, Seiichi Taguchi
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 97 1 205 - 210 2013年01月 [査読有り][通常論文]
     
    (R)-3-hydroxybutyrate [(R)-3HB] is a useful precursor in the synthesis of value-added chiral compounds such as antibiotics and vitamins. Typically, (R)-3HB has been microbially produced from sugars via modified (R)-3HB-polymer-synthesizing pathways in which acetyl CoA is converted into (R)-3-hydroxybutyryl-coenzyme A [(R)-3HB-CoA] by beta-ketothiolase (PhaA) and acetoacetyl CoA reductase (PhaB). (R)-3HB-CoA is hydrolyzed into (R)-3HB by modifying enzymes or undergoes degradation of the polymerized product. In the present study, we constructed a new (R)-3HB-generating pathway from glucose by using propionyl CoA transferase (PCT). This pathway was designed to excrete (R)-3HB by means of a PCT-catalyzed reaction coupled with regeneration of acetyl CoA, the starting substance for synthesizing (R)-3HB-CoA. Considering the equilibrium reaction of PCT, the PCT-catalyzed (R)-3HB production would be expected to be facilitated by the addition of acetate since it acts as an acceptor of CoA. As expected, the engineered Escherichia coli harboring the phaAB and pct genes produced 1.0 g L-1 (R)-3HB from glucose, and with the addition of acetate into the medium, the concentration was increased up to 5.2 g L-1, with a productivity of 0.22 g L-1 h(-1). The effectiveness of the extracellularly added acetate was evaluated by monitoring the conversion of C-13 carbonyl carbon-labeled acetate into (R)-3HB using gas chromatography/mass spectrometry. The enantiopurity of (R)-3HB was determined to be 99.2% using chiral liquid chromatography. These results demonstrate that the PCT pathway achieved a rapid co-conversion of glucose and acetate into (R)-3HB.
  • John Masani Nduko, Ken'ichiro Matsumoto, Toshihiko Ooi, Seiichi Taguchi
    METABOLIC ENGINEERING 15 159 - 166 2013年01月 [査読有り][通常論文]
     
    Xylose, which is a major constituent of lignocellulosic biomass, was utilized for the production of poly(lactate-co-3-hydroxybutyrate) [P(LA-co-3HB)], having transparent and flexible properties. The recombinant Escherichia coli JW0885 (pflA(-)) expressing LA-polymerizing enzyme (LPE) and monomer supplying enzymes grown on xylose produced a copolymer having a higher LA fraction (34 mol%) than that grown on glucose (26 mol%). This benefit of xylose was further enhanced by combining it with an evolved LPE (ST/FS/QK), achieving a copolymer production having 60 mol% LA from xylose, while glucose gave a 47 mol% LA under the same condition. The overall carbon yields from the sugars to the polymer were similar for xylose and glucose, but the ratio of the LA and 3HB units in the copolymer was different. Notably, the P(LA-co-3HB) yield from xylose (7.3 g l(-1)) was remarkably higher than that of P(3HB) (4.1 g l(-1)), indicating P(LA-co-3HB) as a potent target for xylose utilization. (C) 2012 Elsevier Inc. All rights reserved.
  • Toshihiko Ooi, KeN'Ichiro Matsumoto, Ryousuke Kadoya, Seiichi Taguchi
    Kobunshi Ronbunshu 70 12 675 - 683 2013年 [査読無し][通常論文]
     
    This review touches on recent trials for producing microbial polyhydroxyalkanoates (PHAs), which are used as a bio-based plastic, from renewable and non-edible lignocellulosic biomass. Lignocellulose is composed of cellulose, hemicellulose and lignin, which form a persistent complex. Thus, for the efficient saccharification of lignocellulose, the physical/chemical processes for removing lignin and unstiffening cellulose fibers are required. The obtained sugar is typically a mixture of glucose and xylose, and contains a certain inpurity derived from lignocellulose and/or byproduct generated during pretreatment and subsequent saccharification processes. The microbes used for PHA production need to utilize the mixed sugar and to be resistant to the impurities. This review introduces several examples for addressing this issue. Moreover, an important direction is to design the polymer with better properties. Metabolic and enzyme engineering are powerful tools to biosynthesize various useful polymers from nonrelated sugar carbon sources. In particular, microbial production of lactate-based polymer from xylose is a potent platform. ©2013, The Society of Polymer Science,.
  • Bioconversion of the hemicellulosic sugar, xylose, into lactate-based polyesters using recombinant Escherichia coli
    Nduko John M, Matsumoto Ken'ichiro, Ooi Toshihiko, Taguchi Seiichi
    ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 243 2012年03月25日 [査読有り][通常論文]
  • Toshihiko Ooi, Daiki Ogata, Ken'ichiro Matsumoto, Gen Nakamura, Jian Yu, Min Yao, Masaya Kitamura, Seiichi Taguchi
    JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC 74 3-4 204 - 208 2012年02月 [査読無し][通常論文]
     
    The flavin selectivity of the flavoenzymes is considered to be very strict in terms of the functional expression of the enzyme. However, we found that an FMN-dependent azoreductase from Bacillus sp. B29 exhibited up to 60% of the activity of native AzrA harboring FMN upon the addition of a FAD cofactor. The FAD binding to the apo-form of AzrA was identified by spectrophotometric analysis, and the bound FAD was stably retained in the enzyme molecule without degradation to FMN. On the other hand, no effect of riboflavin on the activity of AzrA was detected and there was no obvious quenching of riboflavin detected with the addition of apoAzrA. By a docking simulation of FAD into the structure of a homolog of AzrA (AzoR from Escherichia coli), we created a FAD-binding model. Taking all of these results together, it is proposed that the isoalloxazine ring of FAD localizes at the same site and plays the same role as that of FMN in AzrA. (C) 2011 Elsevier B.V. All rights reserved.
  • John Masani Nduko, Wakako Suzuki, Ken'ichiro Matsumoto, Hirokazu Kobayashi, Toshihiko Ooi, Atsushi Fukuoka, Seiichi Taguchi
    JOURNAL OF BIOSCIENCE AND BIOENGINEERING 113 1 70 - 72 2012年01月 [査読有り][通常論文]
     
    Poly[3-hydroxybutyrate-co-3-hydroxyvalerate(3HV)] was produced in recombinant Escherichia coli LS5218 from ruthenium-catalyzed cellulose hydrolysate and propionate. The strain was found to be resistant to 5-hydroxymethylfurfural (5-HMF), which is a major inhibitory byproduct generated in the cellulose hydrolysis reaction. The 3HV fraction was successfully regulated in the range of 5.6-40 mol%. (C) 2011, The Society for Biotechnology, Japan. All rights reserved.
  • Ken'ichiro Matsumoto, Yuichi Mukai, Daiki Ogata, Fumi Shozui, John Masani Nduko, Seiichi Taguchi, Toshihiko Ooi
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 86 5 1431 - 1438 2010年05月 [査読有り][通常論文]
     
    The gene encoding an FMN-dependent NADH azoreductase, AzrG, from thermophilic Geobacillus stearothermophilus was cloned and functionally expressed in recombinant Escherichia coli. Purified recombinant AzrG is a homodimer of 23 kDa and bore FMN as a flavin cofactor. The optimal temperature of AzrG was 85 A degrees C for the degradation of Methyl Red (MR). AzrG remained active for 1 h at 65 A degrees C and for 1 month at 30 A degrees C, demonstrating both superior thermostability and long-term stability of the enzyme. AzrG efficiently decolorized MR, Ethyl Red at 30 A degrees C. Furthermore, the enzyme exhibited a wide-range of degrading activity towards several tenacious azo dyes, such as Acid Red 88, Orange I, and Congo Red. These results suggested the sustainable utilization of G. stearothermophilus as an azo-degrading strain for AzrG carrying whole-cell wastewater treatments for azo pollutants under high temperature conditions.
  • Daiki Ogata, Toshihiko Ooi, Takaaki Fujiwara, Seiichi Taguchi, Isao Tanaka, Min Yao
    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS 66 Pt 5 503 - 505 2010年05月 [査読有り][通常論文]
     
    Azoreductases from Bacillus sp. B29 are NADH-dependent flavoenzymes which contain a flavin mononucleotide (FMN) as a prosthetic group and exist as homodimers composed of 23 kDa subunits. These enzymes catalyze the reductive degradation of various azo compounds by a ping-pong mechanism. In order to determine the structure-function relationship of the azo-dye reduction mechanism, an X-ray crystallographic study of azoreductases was performed. Selenomethionine-labelled AzrA (SeMet-AzrA) and AzrC were crystallized by the hanging-drop vapour-diffusion method. A crystal of SeMet-AzrA diffracted to 2.0 angstrom resolution and was determined to belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 56.9, b = 69.0, c = 105.4 angstrom. The native crystals of AzrC belonged to space group C2, with unit-cell parameters a = 192.0, b = 56.6, c = 105.5 angstrom, beta = 115.7 degrees, and diffracted to 2.21 angstrom resolution.
  • Ken'ichiro Matsumoto, Yoshitake Orikasa, Kenta Ichinohe, Shigeki Hashimoto, Toshihiko Ooi, Seiichi Taguchi
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 395 1 7 - 10 2010年04月 [査読有り][通常論文]
     
    Contributing factors for the antimicrobial activity enhancement of N-terminally engineered mutants of cell-penetrating apidaecins were analyzed based on their cell-penetration efficiency. The flow cytometric analysis of the engineered apidaecins labeled with carboxyfluorescein (FAM) revealed their enhanced cell-penetrating efficiencies into Escherichia coli that should be one of key factors causing the enhanced antimicrobial activity. It is noteworthy that, for one mutant, the enhancement in antimicrobial activity (18-fold higher than wild type) was greater than that of cell penetration (5.9-fold), suggesting that the N-terminal mutation may reinforce both interaction with unidentified intracellular target(s) and cell-penetration efficiency. (C) 2010 Elsevier Inc. All rights reserved.
  • Toshihiko Ooi, Hirokazu Sato, Ken'ichiro Matsumoto, Seiichi Taguchi
    PROTEIN EXPRESSION AND PURIFICATION 67 2 126 - 131 2009年10月 [査読有り][通常論文]
     
    To characterize an exo-beta-1,3-glucanase (ExgP) of an isolated fungal strain with high laminarin degradation activity, identified as Penicillium sp. KH10, heterologous secretory expression of the ExgP was performed in Aspergillus oryzae. Deduced amino acid sequence of the exgP gene possibly consisted of 989 amino acids which showed high sequence similarity to those of fungal exo-beta-1,3-glucanases belonging to the glycoside hydrolase (GH) family 55. Notably, the purified recombinant ExgP showed a single protein peak in the native state (by gel-permeation chromatographic analysis), but showed two protein bands in the denatured state (by SDS-polyacrylamide gel electrophoresis). These two polypeptides exhibited activity in a coexisting state even under reducing conditions, suggesting that non-covalent association of both polypeptides took place. Taken together with the nucleotide sequence information, the ExgP precursor (104 kDa) would be proteolytically processed (cleaved) to generate two protein fragments (42 and 47 kDa) and the processed products (polypeptide fragments) would be assembled each other by a non-covalent interaction. Moreover, one of the matured ExgP polypeptides was N-glycosylated by the post-translational modi. cation. (C) 2009 Elsevier Inc. All rights reserved.
  • Yoshitake Orikasa, Kenta Ichinohe, Junki Saito, Shigeki Hashimoto, Ken'ichiro Matsumoto, Toshihiko Ooi, Seiichi Taguchi
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 73 7 1683 - 1684 2009年07月 [査読有り][通常論文]
     
    The chemically modified thanatins with the methyll group (CH3), ethyl group (C2H5), and normal-octyl group (C8H17) at the side-chain of cysteine residues were synthesized. The octyl group modified form exhibited 8-fold higher antimicrobial activity against Micrococcus luteus than wild type thanatin. It was found that there was an equilateral correlation between antimicrobial activity and side-chain hydrophobicity at the cysteine residues in thanatin.
  • Toshihiko Ooi, Takeshi Shibata, Ken'ichiro Matsumoto, Shinichi Kinoshita, Seiichi Taguchi
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 73 5 1209 - 1211 2009年05月 [査読有り][通常論文]
     
    We cloned and expressed two genes encoding azoreductase homologes, AzrB and AzrC, from Bacillus sp. B29. Purified recombinant AzrB and AzrC were homodimers with 23kDa identical subunits, and were flavoproteins. NADH was preferred as electron donors for both azoreductases. The azoreductases showed optimal activities at 70 degrees C (AzrB) and 55 degrees C (AzrC), and retained activities up to 55 degrees C (AzrB) and 50 degrees C (AzrC) after incubation for 1 h. Other enzymatic properties, including the substrate specificities of both azoreductases, were also investigated.
  • Sung-Jin Jo, Ken'ichiro Matsumoto, Chean Ring Leong, Toshihiko Ooi, Seiichi Taguchi
    JOURNAL OF BIOSCIENCE AND BIOENGINEERING 104 6 457 - 463 2007年12月 [査読有り][通常論文]
     
    In our previous study, a system for producing poly(3-hydroxybutyrate) [P(3HB)] was established by introducing a polyhydroxyalkanoate (PHA) biosynthetic gene operon (phaCAB(Re)) derived from Ralstonia eutropha into Corynebacterium glutamicum. In this study, two experimental strategies have been applied to improve P(3HB) production in recombinant C glutamicum. One is a codon optimization of the N-terminal-coding region of the PHA synthase (PhaC(Re)) gene focusing on the codon usage preference for the translation system of C. glutamicum. The other is the replacement of wild-type phaC,, with a modified gene encoding a mutation of Gly4Asp (G4D), which enhanced the production of PhaCRe and P(3HB) in Escherichia coli. The introduction of these engineered PHA synthase genes into C glutamicum enhanced the production of PhaC(Re) and P(3HB). Interestingly, we found that these gene modifications also caused increases in the concentration of the translation products of the genes encoding monomer-supplying enzymes, beta-ketothiolase (PhaA(Re)) and acetoacetyl-CoA reductase (PhaB(Re)). This finding prompted us to carry out a gene dosage of phaAB(Re) for a double plasmid system, and the highest production (52.5 wt%) of P(3HB) was finally achieved by combining the gene dosage of phaAB(Re) with codon optimization. The molecular weight of P(3HB) was also increased by approximately 2-fold, as was P(3HB) content. Microscopic observation revealed that the volume of the cells accumulating P(31113) was increased by more than 4-fold compared with the non -P(3HB)-accumulating cells without filamentous morphologenesis observed in E. coli.
  • Toshihiko Ooi, Takeshi Shibata, Reiko Sato, Hiroaki Ohno, Shinichi Kinoshita, Tran Linh Thuoc, Seiichi Taguchi
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 75 2 377 - 386 2007年05月 [査読有り][通常論文]
     
    The gene coding for an azoreductase, designated as an azrA, was cloned by polymerase chain reaction amplification from the genomic DNA of Bacillus sp. strain B29 isolated from soil. The azrA encoded a protein of 208 amino acids with calculated molecular mass of 22,766 Da. The enzyme was heterologously expressed in Escherichia coli with a strong band of 23 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Purified recombinant AzrA was a homodimer with a native molecular mass of 48 kDa containing two molecules of flavin mononucleotide (FMN; oxidized). This activity was oxygen insensitive and was nicotinamide adenine dinucleotide (reduced form; NADH) dependent. Recombinant AzrA exhibited a broad pH stability between 6 and 10 with a temperature optimum of 60-80 degrees C. The enzyme cleaved the model azo compound of methyl red [MR, 4'-(dimethylamino)-azobenzene-2-carboxylic acid] into 2-aminobenzoic acid and N, N'-dimethyl-p-phenylenediamine by ping-pong mechanism. The enzyme was not only able to decolorize MR but also able to decolorize sulfonated azo dyes such as Orange I and Acid Red 88.
  • Sung-Jin Jo, Michihisa Maeda, Toshihiko Ooi, Seiichi Taguchi
    JOURNAL OF BIOSCIENCE AND BIOENGINEERING 102 3 233 - 236 2006年09月 [査読有り][通常論文]
     
    A biosynthetic pathway for poly(3-hydroxybutyrate) [P(3HB)] production by Corynebacterium glutamicum was developed by introducing the phbCAB operon derived from Ralstonia eutropha. P(3HB) synthase activity was detected in this recombinant C glutamicum carrying a cell surface protein gene promoter. Intracellular P(3HB) was microscopically observed as inclusion granules and its content was calculated to be 22.5% (w/w) with a number average molecular weight of 2.1 x 10(5) and a polydispersity of 1.63.
  • K Okuno, M Yabuta, T Ooi, S Kinoshita
    APPLIED AND ENVIRONMENTAL MICROBIOLOGY 70 1 76 - 86 2004年01月 [査読有り][通常論文]
     
    Escherichia coli outer-membrane endoprotease OmpT has suitable properties for processing fusion proteins to produce peptides and proteins. However, utilization of this protease for such production has been restricted due to its generally low cleavage efficiency at Arg (or Lys)-Xaa, where Xaa is a nonbasic N-terminal amino acid of a target polypeptide. The objective of this study was to generate a specific and efficient OmpT protease and to utilize it as a processing enzyme for producing various peptides and proteins by converting its substrate specificity. Since OmpT Asp(97) is proposed to interact with the P1' amino acid of its substrates, OmpT variants with variations at Asp97 were constructed by replacing this amino acid with 19 natural amino acids to alter the cleavage specificity at Arg (P1)-Xaa (P1'). The variant OmpT that had a methionine at this position, but not the wild-type OmpT, efficiently cleaved a fusion protein containing the amino acid sequence -Arg-Arg-ArgAla-Arg down arrow motilin, in which motilin is a model peptide with a phenylalanine at the N terminus. The OmpT variants with leucine and histidine at position 97 were useful in releasing human adrenocorticotropic hormone (1-24) (serine at the N terminus) and human calcitonin precursor (cysteine at the N terminus), respectively, from fusion proteins. Motilin was produced by this method and was purified up to 99.0% by two chromatographic steps; the yield was 160 mg/liter of culture. Our novel method in which the OmpT variants are used could be employed for production of various peptides and proteins.
  • K Okuno, M Yabuta, K Ohsuye, T Ooi, S Kinoshita
    BIOTECHNOLOGY AND APPLIED BIOCHEMISTRY 36 Pt 2 77 - 84 2002年10月 [査読有り][通常論文]
     
    The Escherichia coli outer-membrane endoprotease OmpT mainly cleaves peptide bonds between consecutive basic amino acids. The effect of adjacent residues on cleavage efficiency is currently unknown, except at positions P2 and P2'. Therefore we investigated the effects of amino acid residues upstream of the cleavage site on the ability of OmpT to cleave efficiently a fusion protein carrying human glucagon-like peptide-1 (7-37) in 4 M urea. The P1-P10 residues were replaced by Ala and each substrate was subjected to OmpT digestion. The replacement of Arg residue at P1 blocked the cleavage due to the loss of the cleavage site, and the replacement of Arg residue at P4 maximally reduced the cleavage rate. Conversely, cleavage efficiency increased on replacing Glu at P6. Substitution of the residues at P4 and P6 with several different amino acids showed that OmpT preferred basic residues at these positions, whereas acidic residues had a negative effect. This was also shown to be true with synthetic decapeptide substrates in the absence of urea. The k(cat)/K-m ratio increased with basic residues at P4 or P6, mainly due to a lower 14 rather than an increase in k(cat). On the basis of these findings, we prepared a fusion protein carrying human atrial natriuretic peptide (ANP), a drug for acute congestive heart failure. OmpT released mature ANP from the E. coli-expressed fusion protein. As expected, the introduction of an Arg residue at P4 and P6 enhanced the release of ANP.
  • K Okuno, M Yabuta, K Kawanishi, K Ohsuye, T Ooi, S Kinoshita
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 66 1 127 - 134 2002年01月 [査読有り][通常論文]
     
    Though OmpT has been reported to mainly cleave the peptide bond between consecutive basic amino acids, we identified more precise substrate specificity by using a series of modified substrates, termed PRX fusion proteins, consisting of 184 residues. The cleavage site of the substrate PRR was Arg(140)-Arg(141) and the modified substrates PRX substituted all 19 natural amino acids at the P1' site instead of Arg(141). OmpT under denaturing conditions (in the presence of 4 m urea) cleaved not only between two consecutive basic amino acids but also at the carboxyl side of Arg(140) except for the Arg(140)-ASP(141,) -Glu(141), and -Pro(141) pairs. In addition to Arg(140) at the PI site, similar results were obtained when Lys(140) was substituted into the P1 site. In the absence of urea, an aspartic acid residue at the P1' site was unfavorable for OmpT cleavage of synthetic decapeptides, the enzyme showed a preference for a dibasic site.
  • J. Kraiwattanapong, T. Ooi, S. Kinoshita, I. Sugimura, T. Sawabe, Y. Ezura
    World Journal of Microbiology and Biotechnology 16 3 219 - 224 2000年 [査読無し][通常論文]
     
    Sixteen alginate lyases whose primary sequences have been reported were compared, and classified into the following three groups on the basis of the identity of their primary sequences. Strong homology (> 50%): A-AlgL, A-AlgL*, P-AlgL, P-AlgL*, and AlgA weak homology (> 20%): ALY, AlxM, P-Aly, K-Aly, AlyPG, AlgVGI, AlgVGII, and AlgVGIII little homology (< 20%): ALYII, Al-III, and AlgVMI. Using hydrophobic cluster analysis (HCA), a secondary structure prediction method, the sixteen alginate lyases were placed into the following classes. Class 1: AlgA, A-AlgL, A-AlgL*, P-AlgL, and P-AIgL* Class 2: AlgVMI and Al-III Class 3: ALY and AlxM Class 4A: ALYII, K-Aly, P-Aly, and AlyPG Class 4B: AlgVGI and AlgVGII Class 5: AlgVGIII, which is put in a class of its own, because it is unlike any of the other alginate lyases.
  • Yasumitsu Miyamoto, Toshihiko Ooi, Shinnichi Kinoshita
    Biotechnology Letters 22 5 427 - 430 2000年 [査読無し][通常論文]
     
    Pseudomonas sp. LS 13-1 was isolated as a producer of lactobionic acid from whey and when grown with 207 g whey l-1 (150 g lactose l-1 equivalent) and three intermittent additions of 69 g whey l-1 (50 g lactose l-1 equivalent) in a fed-batch culture at pH 5.5 in a 2-l jar fermenter, it produced 175 g lactobionic acid l-1 after 180 h. In a lactose medium it produced 240 lactobionic acid l-1 from a total of 300 g lactose l-1 after 155 h. With the addition of 20 CaCO3 l-1 instead of pH control, 290 g lactobionic acid l-1 was produced in the lactose medium after 155 h with a yield of higher than 90% (mon mol-1).
  • A Ohnishi, T Ooi, S Kinoshita, H Tomatsuri, K Umeda, S Ueda, Y Hata, M Arai
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 63 12 2157 - 2162 1999年12月 [査読無し][通常論文]
     
    Four acidic amino acid residues, Asp97, Asp101, Glu118, and Glu202, were located in the cleft from the Xray crystallographic analysis of FI-CMCase, endo-1,4-beta-glucanase (EC: 3.2.1.4) of Aspergillus aculeatus No. F-50. To identify the catalytic residues of the FI-CMCase, these residues were mutated to Glu or Ser from Asp97 and Asp101, and to Asp or Ser from Glu118 and Glu202 by site-directed mutagenesis, and totally 8 single mutant enzymes experssed in Escherichia coli were prepared: D97E, D97S, D101E, D101S, E118D, E118S, E202D, and E202S. Mutant enzymes E118S and E202S were not shown to have any detectable activity. Kinetic parameters of other mutant enzymes were measured after purification. The Km of mutant enzymes were not much different from that of wild type FI-CMCase, while the Vmax of mutant enzymes D97E, D97S, D101E, D101S, E118D, and D202E were much decreased to 1/50, 1/20, 1/4000, 1/2000, 1/800, and 1/1600 of the wild type FI-CMCase, respectively. From these results we concluded that Glu118 and Glu202 were most probable candidates for a catalytic pair of acidic amino acids in FI-CMCase.
  • T. Ooi, K. Morotomi, S. Kinoshita, J. Honda, S. Ueda, M. Arai
    Biotechnology Letters 21 9 735 - 739 1999年 [査読無し][通常論文]
     
    The 5'-upstream non-coding region of an FI-carboxymethyl cellulase (CMCase) gene of Aspergillus aculeatus No. F-50 was obtained and sequenced up to -777 nucleotide in pCMG23. The 5'-upstream region of different lengths were constructed and fused to a reporter gene (Escherichia coli lacZ) in pAN923-42BD, and the resulted constructs were introduced into the A. nidulans. The β-galactosidase activities of the transformant with 5'-upstream fragment of larger than 319 bp were expressed by the induction, but that of 109 bp drastically decreased to the basal level. This suggests that the region between -109 and -319 of the 5'-upstream non-coding region is involved in the regulation of the FI-CMCase expression.
  • J. Kraiwattanapong, K. Motomura, T. Ooi, S. Kinoshita
    World Journal of Microbiology and Biotechnology 15 117 - 122 1999年01月01日 [査読無し][通常論文]
     
    An alginate lyase named ALYII was purified to homogeneity from Escherichia coli JM109 carrying a recombinant plasmid, pJK26 harbouring the alyII gene from Pseudomonas sp. OS-ALG-9 by column chromatography with DEAE-cellulose, CM-Sephadex C-50, butyl-Toyopearl 650M and isoelectric focusing. The molecular size of the purified ALYII was estimated to be 79 kDa by SDS-PAGE and its pI was 8.3. The enzyme was most active at pH 7.0 and 30 °C. Its activity was completely inhibited by Hg2+. The enzyme was poly β-D-1, 4-mannuronate-specific rather than β-D-1, 4-guluronate-specific and it showed a promotion effect in alginate degradation by combination with ALY, an another poly β-D-1, 4-mannuronate-specific alginate lyase from the same strain.
  • C. Khanongnuch, T. Ooi, S. Kinoshita
    World Journal of Microbiology and Biotechnology 15 221 - 228 1999年01月01日 [査読無し][通常論文]
     
    The two genes for β-mannanase and cellulase of Bacillus sp. 5H have been cloned in Escherichia coli JM 109 by a shotgun method, though the cellulase gene was not expressed in Bacillus sp. 5H. The nucleotide sequences of the β-mannanase gene and the cellulase gene revealed open reading frames of 1,086 and 1,503 base pairs, respectively, coding for a proteins of M(r) 40,803 Da (β-mannanase) and 55,420 Da (cellulase). The deduced primary structure of β-mannanase comprised 362 amino acids which had a mature protein of 336 amino acids and a signal peptide of 26 amino acids and that of cellulase comprised 501 amino acid residues.
  • Chartchai Khanongnuch, Saisamorn Lumyong, Toshihiko Ooi, Shinichi Kinoshita
    Biotechnology Letters 21 1 61 - 63 1999年 [査読無し][通常論文]
     
    A newly isolated strain of Bacillus subtilis produced β-mannanase when cultivated in a medium containing either locust bean gum, konjac mannan or guar gum as a sole carbon source. In contrast, xylanase was produced only when oat spelt xylan or wheat bran was used as a carbon source. The culture supernatant, which contained both β-mannanase and xylanase, was used to biobleach crude paper pulp to 50% gain in brightness.
  • Chartchai Khanongnuch, Kouhei Asada, Hideo Tsuruga, Toshihiko Ooi, Shinichi Kinoshita, Saisamorn Lumyong
    Journal of Fermentation and Bioengineering 86 5 461 - 466 1998年 [査読無し][通常論文]
     
    A bacterial strain, 5H was isolated as a utilizer of locust bean gum (LBG) from soil in Chiang Mai, Thailand, and identified as Bacillus subtilis. Strain 5H produced β-mannanase and xylanase, which were purified to homogeneity from the culture filtrate by ion exchange chromatography and gel filtration. The molecular weights of β-mannanase and xylanase were found to be 37,000 and 26,000 by sodium dodecyl sulfonate polyacrylamide gel electrophoresis, respectively. The enzymatic properties of both were determined. Mannanase hydrolyzed LBG and konjak mannan endwisely, to a final hydrolytic degree of 15% and 21%, respectively, and the main products of both were disaccharides. Xylanase hydrolyzed larch wood xylan and oat spelt xylan, to a final hydrolytic degree of 19% and 38%, respectively, and produced mainly disaccharides. This culture filtrate (4.9 units/ml β-mannanase and 3.2 units/ml xylanase), a mixture of purified β-mannanase (4.9 units/ml) and xylanase (3.2 units/ml), and purified xylanase (3.2 units/ml) bleached crude paper pulp from 30% to 38% brightness, and xylanase was found to be more effective for paper-bleaching than β-mannanase. | A bacterial strain, 5H was isolated as a utilizer of locust bean gum (LBG) from soil in Chiang Mai, Thailand, and identified as Bacillus subtilis. Strain 5H produced β-mannanase and xylanase, which were purified to homogeneity from the culture filtrate by ion exchange chromatography and gel filtration. The molecular weights of β-mannanase and xylanase were found to be 37,000 and 26,000 by sodium dodecyl sulfonate polyacrylamide gel electrophoresis, respectively. The enzymatic properties of both were determined. Mannanase hydrolyzed LBG and konjak mannan endwisely, to a final hydrolytic degree of 15% and 21%, respectively, and the main products of both were disaccharides. Xylanase hydrolyzed larch wood xylan and oat spelt xylan, to a final hydrolytic degree of 19% and 38%, respectively, and produced mainly disaccharides. This culture filtrate (4.9 units/ml β-mannanase and 3.2 units/ml xylanase), a mixture of purified β-mannanase (4.9 units/ml) and xylanase (3.2 units/ml), and purified xylanase (3.2 units/ml) bleached crude paper pulp from 30% to 38% brightness, and xylanase was found to be more effective for paper-bleaching than β-mannanase.
  • J Kraiwattanapong, T Ooi, S Kinoshita
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 61 11 1853 - 1857 1997年11月 [査読無し][通常論文]
     
    Pseudomonas sp. OS-ALG-9 produces several kinds of alginate-degrading enzymes both intra-and extracellularly. As a second alginate lyase of this bacterium, the gene encoding alyII has been cloned in Escherichia coli JM109 by shotgun techniques and then sequenced. The alyII gene has an open reading frame of 2141bp encoding 713 amino acid residues with a calculated molecular mass of 79,803 Da. The deduced amino acid sequence did not show any extensive similarity with those of other known alginate lyases, however, hydrophobic cluster analysis showed that alyII belonged to class 3 of alginate lyases. The alginate lyase from E. coli harboring the alyII gene showed a single active band, which coincided with one of four major alginate lyases from the crude cell extracts of Pseudomonas sp. OS-ALG-9 on a zymogram.
  • K Nakamiya, T Ooi, S Kinoshita
    JOURNAL OF FERMENTATION AND BIOENGINEERING 84 3 213 - 218 1997年 [査読無し][通常論文]
     
    The hydroquinone peroxidase of the lignin decolorizing bacterium, Azotobacter beijerinckii HM121 degraded 1 g/l of polyacrylamide (M-r 2,000,000) to small molecules (M-r less than one thousand) in 30 min in the presence of 5 mM hydrogen peroxide and 5 mu M tetramethylhydroquinone. The two reaction products were purified, and identified as 1-dodecene-2, 4, 6, 8, 10-pentacarboxyamide and 1-hexadecene-2, 4, 6, 8, 10, 12, 14-heptacarboxyamide comprising 5 and 7 acrylamide units, respectively. The enzyme also degraded 1 g/l of polyacrylic acid (M-r 450,000) in 1 h, 1 g/l of polyethylene glycol (M-r 4,000,000) in 1 h, and 1 g/l of polyvinyl alcohol (M-r 88,000) for 20 h. The degradation rate decreased with a decrease in the degree of polymerization of the polyethylene glycol from M-r 20,000 to 400. In addition 1 g/l of polyethylene glycol (M-r 4,000,000) was degraded in the absence of tetramethylhydroquinone, although it took 20 h.
  • K Nakamiya, T Ooi, S Kinoshita
    JOURNAL OF FERMENTATION AND BIOENGINEERING 84 1 14 - 21 1997年 [査読無し][通常論文]
     
    In order to purify a lignin-degrading enzyme from the bacterium Azotobacter beijerinckii HM121, which rapidly decolorizes lignin, the enzyme activity was monitored by observing the hydroquinone oxidizing activity in the presence of hydrogen peroxide. The activity was bound to the membrane and was solubilized by treating disrupted cells with 0.1% Triton X-100. The enzyme was purified to homogeneity by ammonium sulfate fractionation, hydrophobic chromatography on butyl-Toyopearl 650M, and gel filtration on Toyopearl HW-55F. The relative molecular mass of the enzyme was determined to be 59,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and 55,000 by gel filtration on Toyopearl HW-55F, suggesting it is a monomeric enzyme. The purified enzyme exhibited the maximal activity for hydroquinone oxidation at pH 7.0 and 50 degrees C and was stable up to 70 degrees C for 1 h. The optimum concentration of hydrogen peroxide was 6.0 mM. The enzyme was active toward hydroquinone, vanillin, catechol, caffeic acid, p-phenylenediamine, pyrogallol, methylhydroquinone, and dimethylhydroquinane. Based on these results the enzyme was named hydroquinone peroxidase. The enzyme also decolorized and degraded lignin. Spectral analysis revealed that this peroxidase did not hare iron-heme. Inductively coupled plasma analysis showed the presence of equimolar manganese. The enzyme activity was inhibited by 1 mM EDTA and by 1 mM 1,10-phenanthroline; the activity of the EDTA-inactivated enzyme was restored by the addition of manganese ions. However, the enzyme activity did not require exogenous manganese ions and was not promoted by the addition of manganese. The peroxidase oxidized Mn(II) to Mn(III) in the presence of hydrogen peroxide. The activity was promoted 4 fold by the addition of 0.1 mM ZnCl2. The enzyme formed a hydroxy radical from hydrogen peroxide as an active oxygen molecule. From the above results, the hydroquinone peroxidase was judged to be a new type of lignin-degrading enzyme.
  • S Matsuda, Y Kawanami, H Takeda, T Ooi, S Kinoshita
    JOURNAL OF FERMENTATION AND BIOENGINEERING 83 6 593 - 595 1997年 [査読無し][通常論文]
     
    Bacterial strain HU-M1 was isolated as a mutan-degrading microbe from soil and identified as Bacillus circulans. From an HU-M1 culture supernatant mutanase was purified 260-fold by ammonium sulfate fractionation and DEAE-Toyopearl 650M (twice), Toyopearl HW55-F, and Butyl-Toyopearl 650M column chromatography with an activity recovery of 10%. The M-r of the mutanae is 160,000 (monomer). The optimal pH and temperature for the mutanase are pH 6.9 and 50 degrees C for a 10-min reaction, The mutanase is stable at up to 40 degrees C for 60-min and at pH 6.0 to 11.0 at 4 degrees C for 48 h, and logarithmically inactivated at 54 degrees C with a half life of 2.6 min. Mutan hydrolysates produced by the enzyme had di-, tri-, and tetramer of glucose and the final hydrolysis was 38%.
  • K Nakamiya, G Sakasita, T Ooi, S Kinoshita
    JOURNAL OF FERMENTATION AND BIOENGINEERING 84 5 480 - 482 1997年 [査読無し][通常論文]
     
    The hydroquinone peroxidase of a lignin decolorizing bacterium, Azotobacter beijerinckii HM121 degraded a water-insoluble synthetic polymer, polystyrene, in a two-phase system (dichloromethane-water), in which it was degraded in dichloromethane to small water-soluble molecules in a short time in the presence of hydrogen peroxide and tetramethylhydroquinone. Two grams per liter of polystyrene (M-r 935,000) in the dichloromethane phase was completely transferred to the water phase in 5 min by the enzymatic reaction. The degradation products in the water phase were initially detected at R-f 0.6 (M-r 1,000) in thin layer chromatography after reacting for 5 min, and then at R-f 0 (M-r 350) after reacting for 10 min. Another polystyrene (M-r 114,200) was degraded in a similar manner but low molecular weight polystyrene (M-r 760) was degraded and detected at R-f 0 without producing intermediate products at R-f 0.6 after 1-min reaction.
  • Takashi Kawaguchi, Tatsuji Enoki, Shinji Tsurumaki, Jun Ichi Sumitani, Mitsuhiro Ueda, Toshihiko Ooi, Motoo Arai, Motoo Arai
    Gene 173 2 287 - 288 1996年09月 [査読無し][通常論文]
     
    A cDNA was isolated from an Aspergillus aculeatus cDNA library using synthetic oligodeoxyribonucleotide mixtures that corresponded to the internal amino acid (aa) sequence of mature β-glucosidase 1 (BGL1). Analysis of the nucleotide sequence of the cloned cDNA insert revealed a 2580-bp open reading frame (ORF) that encoded a 860-aa protein. The deduced aa sequence of the ORF shared sequence similarity with several BGL from other microorganisms.
  • Yoshiaki Hatakeyama, Hiroyuki Takeda, Toshihiko Ooi, Shinichi Kinoshita
    Journal of Fermentation and Bioengineering 81 6 518 - 523 1996年 [査読無し][通常論文]
     
    β-Fructofuranosidase was purified to homogeneity by a series of column chromatographies on Butyl Toyopearl 650M, DEAE-Sephadex A-50, Toyopearl HW- 55F, and hydroxyapatite with a yield of 4.6% and 1,100-fold purification from the cell extract of Scopulariopsis brevicaulis N-01. The activity was optimal at 40°C and pH 6-9. The enzyme was a homologous dimer protein and the molecular weight of a subunit was 110,000. Kinetic studies were carried out using the purified β-fructofuranosidase. The K(m) and V(max) values for the formation of 1-kestose, nystose, fructosylxyloside, and difructosylxyloside from sucrose, 1-kestose, sucrose and xylose, and fructosylxyloside were respectively determined. Sucrose inhibited nystose formation from 1-kestose, and glucose inhibited the formation of 1-kestose, fructosylxyloside, and difructosylxyloside competitively. The kinetic constants for the hydrolyses of all the above oligosaccharides were determined at lower concentrations, because at higher concentrations the hydrolyses were inhibited. Taking into the consideration the kinetic studies, the preferential production of 1- kestose over nystose and of fructosylxyloside over 1-kestose and difructosylxyloside in fermentations by S. brevicaulis are discussed.
  • H FUJIMOTO, T OOI, SL WANG, T TAKIZAWA, H HIDAKA, S MURAO, A ARAI
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 59 3 538 - 540 1995年03月 [査読無し][通常論文]
     
    Three distinct extracellular xylanases (FIa-, FIb-, and FIII-xylanases) from a culture filtrate of Aspergillus aculeatus No. F-50 were purified to homogeneity by SDS polyacrylamide gel electrophoresis. The molecular weights of FIa-, FIb-, and FIII-xylanases were estimated to be 18,000, 26,000, and 52,000, and their pi values were pH 5.6, 9.0, and 3.8, respectively. The pH optima of xylanase activities were from 4.5 to 5.0. The optimum temperatures for enzyme activities were from 50 degrees C to 70 degrees C. All three xylanases were highly specific for xylan hydrolysis, and they did not cleave xylobiose.
  • K TANAKA, T KAWAGUCHI, Y IMADA, T OOI, M ARAI
    JOURNAL OF FERMENTATION AND BIOENGINEERING 79 3 212 - 216 1995年 [査読無し][通常論文]
     
    Cellobiose phosphorylase was purified 111-fold from a cell extract of Clostridium thermocellum ATCC 27405, with a yield of 31.4%, to electrophoretic and column chromatographic homogeneity. The molecular weight of the enzyme was estimated to be 150,000 by gel filtration and 85,000 by SDS-PAGE, suggesting that it consisted of two identical subunits. It was suggested by spectrophotometric and chemical analyse that the enzyme contained no pyridoxal phosphate. The enzyme was inactivated by N-ethylmaleimide and activated by dithiothreitol, indicating that the exposed thiol group(s) was important for the enzymatic activity. The enzyme could utilize, so far as examined, D-glucose, D-xylose, 2-deoxy-D-glucose, and D-mannose, as accepters of glucose in the synthetic reaction of disaccharides. The enzyme could to a low degree utilize D-arabinose and D-fucose, as accepters.
  • K MINAMIGUCHI, T OOI, T KAWAGUCHI, H OKADA, S MURAO, M ARAI
    JOURNAL OF FERMENTATION AND BIOENGINEERING 79 4 363 - 366 1995年 [査読無し][通常論文]
     
    A cDNA encoding FI-carboxymethylcellulase (FI-CMCase) of the fungus Aspergillus aculeatus was expressed in Saccharomyces cerevisiae under the control of the glyceraldehyde-3-phosphate dehydrogenase gene (GAP) promoter of S. cerevisiae. The transformed cells were able to secrete FI-CMCase efficiently into the culture medium as active enzyme. The recombinant FI-CMCases were observed to be two different enzymes of different molecular mass, one of which corresponded to native FI-CMCase (non-glycosylated FI-CMCase) and the other of which was an N-glycosylated protein (glycosylated FI-CMCase). The recombinant glycosylated FI-CMCase showed a higher thermostability than that of the native enzyme, although the former showed slightly lower activity toward the substrate than the latter.
  • Y HATA, K NATORI, Y KATSUBE, T OOI, M ARAI, H OKADA
    JOURNAL OF MOLECULAR BIOLOGY 241 2 278 - 280 1994年08月 [査読無し][通常論文]
     
    Most fungal cellulases are found in multiple forms varying in size and substrate specificity. Aspergillus aculeatus is known to produce nine cellulolytic enzymes including an endoglucanase (FI CM-cellulase, M(r) = 24,002) as the major component. Single crystals of FI CM-cellulase from Aspergillus aculeatus have been prepared by sitting-drop vapour diffusion using ammonium sulphate as a precipitant. The cellulase crystals belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions of a = 52.79(2) Angstrom, b = 106.40(4) Angstrom and c = 33.15(1) Angstrom. The crystals contain one enzyme molecule per asymmetric unit. They diffract to at least 2.0 Angstrom resolution and are very stable against X-ray irradiation.
  • T OOI, K MINAMIGUCHI, T KAWAGUCHI, H OKADA, S MURAO, M ARAI
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 58 5 954 - 956 1994年05月 [査読無し][通常論文]
     
    As a step to breed a Saccharomyces cerevisiae strain able to produce ethanol directly from cellulose, we combined cDNA for Aspergillus aculeatus FI-CMCase (FI-carboxymethyl cellulase) with the GAP (glyceraldehyde-3-phosphate dehydrogenase) promoter of S. cerevisiae and used the resultant plasmid, pYEC91, to transform S. cerevisiae. The transformed cells produced active FI-CMCase within the cytoplasm. Western-blot analysis following SDS-polyacrylamide gel electrophoresis demonstrated that the cells contained a peptide having the same molecular mass and immunological identity as A. aculeatus FI-CMCase.
  • T OOI, K MINAMIGUCHI, T KAWAGUCHI, H OKADA, S MURAO, M ARAI
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 57 11 1960 - 1961 1993年11月 [査読無し][通常論文]
     
    FI-CMCase cDNA of Aspergillus aculeatus was expressed in Escherichia coli by using the tac promoter of E. coli. Transformants of E. coli harboring a plasmid pHEM06 containing mature form FI-CMCase cDNA produced FI-CMCase in the cytoplasm of the cells. The enzyme from E. coli cells was purified to yield 56% and it was immunological identical to that of FI-CMCase purified from A. aculeatus.
  • T OOI, A SHINMYO, H OKADA, S HARA, T IKENAKA, S MURAO, M ARAI
    CURRENT GENETICS 18 3 217 - 222 1990年10月 [査読無し][通常論文]
     
    Wa have cloned and characterized the cDNA coding for a major component of cellulase, endoglucanase (FI-CMCase), produced by Aspergillus aculeatus. The cDNA was isolated from a A. aculeatus cDNA library using synthetic oligonuceotide mixtures that correspond to the internal amino acid sequence of the mature FI-CMCase protein. Nucleotide sequence analysis of the cloned cDNA insert revealed a 711 bp open reading frame that encoded a protein of 237 amino acid residues. The primary structure of FI-CMCase deduced from the nucleotide sequence of cDNA agreed with that found by amino acid sequencing of peptide fragments obtained by digestion with several proteinases and cyanogen bromide cleavage. There may be a signal peptide sequence of 16 amino acid residues at the N-terminus. The molecular mass of the mature protein calculated from the cDNA is 24002 daltons, which compares favorably with molecular mass estimates of purified FI-CMCase obtained from SDS-PAGE (25000 Da). No distinct homology was found between the amino acid sequence of FI-CMCase and known cellulase sequences of other microorganisms. This study is the first example of cDNA cloning of an endoglucanase from the genus Aspergillus. © 1990 Springer-Verlag.
  • T OOI, A SHINMYO, H OKADA, S MURAO, T KAWAGUCHI, M ARAI
    NUCLEIC ACIDS RESEARCH 18 19 5884 - 5884 1990年10月 [査読無し][通常論文]
  • M ARAI, T OOI, H HAYASHI, S MURAO
    HAKKOKOGAKU KAISHI-JOURNAL OF THE SOCIETY OF FERMENTATION TECHNOLOGY 63 5 427 - 431 1985年 [査読有り][通常論文]

MISC

所属学協会

  • 日本応用糖質科学会   日本生物工学会   日本農芸化学会   

共同研究・競争的資金等の研究課題

  • 文部科学省:科学研究費補助金(基盤研究(C))
    研究期間 : 2014年 -2016年 
    代表者 : 大井 俊彦
  • 文部科学省:科学研究費補助金(基盤研究(C))
    研究期間 : 2011年 -2013年 
    代表者 : 大井 俊彦, 田口 精一, 松本 謙一郎
     
    高機能バイオプラスチックを合成できる反応集積型の「微生物工場」は、バイオマスからのグルコースを選択的に資化して駆動することが出来るが、現状ではバイオマスの糖化プロセスではグルコース以外の不純物の副生は不可避である。その中でも生育阻害物質に対する耐性が弱いことからバイオマス糖化物中に複製されるルルフラールやその関連化合物に対する「微生物工場」の耐性が望まれる。本年度はルテニウム担持触媒を用いたセルロース加水分解物中に含まれるフルフラールやその関連化合物等が微生物工場である大腸菌の生育を阻害するので、これを回避するために、主要な阻害物質である5-メチルフルフラール(5-HMF)は予備実験において大腸菌の生育は阻害するが、ポリマーの生産には大きな影響がないことがわかったので、5-HMFに耐性を持つ大腸菌を探索した。その結果、LS5218株は3g/Lまではほとんど生育に影響しないことがわかった。この5-HMF耐性はセルロース加水分解物中に含まれる5-MF濃度とグルコース濃度からポリマー生産条件での最適グルコース濃度であっても生育に影響しないことから、セルロース加水分解物を炭素源としてPHAの生産を行ったところ、添加したプロピオン酸の濃度に依存した、3-ヒドロキシ吉草酸を含むPHAが得られた。
  • 文部科学省:科学研究費補助金(一般研究(C), 基盤研究(C))
    研究期間 : 1995年 -1996年 
    代表者 : 大井 俊彦
     
    糸状菌A. aculeatus由来のセルラーゼおよび細菌B. pumilus由来のキシラナーゼは進化的にも遠く、両酵素蛋白質間の一次構造上の相同性は低い。しかしながら、それらの立体構造は非常に高い類似性を示す。そこで不溶性糖質加水分解酵素の構造と機能を解析するため、両酵素間のキメラ酵素蛋白質を遺伝子操作技術を用いて作製し、得られたキメラ酵素の立体構造と活性発現の関係に関する基礎的知見を検討することを最終目的としている。平成7年度においては、キメラ酵素の構築とそれらの高発現系について中心的に検討した。平成8年度においては引き続きこれらの精製と性質について検討した。両酵素とも2枚のβ-シートの間にはさまれたクレフトと考えられる領域に活性部位が存在し触媒活性を直接担っていると考えられている。両酵素のα,β領域とβ,β領域を連結するヒンジ部分でそれぞれの領域が分断できる構造のため、キメラ酵素構築のためには両酵素分子のβ,β-領域とα,β-領域とを分断し両者をそれぞれ交換した形で構築した。セルラーゼ[C]およびキシラナゼ[X]の両構造遺伝子上においては、キメラ-1は[X-C-X]、キメラ-2は[C-X-C]となるように各遺伝子の読み取り枠を保存して連結した構造になっている。両キメラ遺伝子を各々保持する各形質転換株の全蛋白質をSDS-PAGEで分析したところ、予想された分子量に相当する蛋白質バンドの存在が確認された。さらに、これら蛋白質は両酵素に対する抗血清と反応することがウェスタンブロッティングにより確認されたことから、両キメラ蛋白質の発現していることが確認された。キメラタンパク質を精製するために、LB培地を用いて培養した後、集菌後菌体を超音波処理にて破砕後、除核酸、硫安沈殿の後、透析し各種カラムクロマトにより抗原・抗体反応を指標にして精製を行った。精製したキメラ酵素蛋白質は電気泳動的に単一であった。アグリコンとして4-メチルウンベルフェリル(4-MU)を持った市販の蛍光基質に対する酵素活性を調べたところ、セロビオース、セロトリオース、キシロースの基質に対しては活性は検出限界以下であった。
  • 文部科学省:科学研究費補助金(奨励研究(A))
    研究期間 : 1994年 -1994年 
    代表者 : 大井 俊彦
     
    糸状菌A.aculeatus由来のセルラーゼおよび細菌B.pumilus由来のキシラナーゼは進化的にも遠いと考えられ、両酵素蛋白質間の一次構造上の相同性は低い。しかしながら、それらの立体構造はβ-サンドイッチ構造と呼ぶホールディング形式を持ち非常に高い類似性を示す。このことは、セルロース関連の不溶性糖質加水分解酵素の構造と機能を解明する上で、、必須と考えられる酵素蛋白質分子のホールディングと酵素機能の関係を理解するための非常に良い題材であると考えられる。そこで不溶性糖質加水分解酵素の構造と機能を蛋白質工学的手法により解析するため、両酵素間のキメラ酵素蛋白質を遺伝子操作技術を用いて作製し、得られたキメラ酵素の立体構造と活性発現の関係に関する基礎的知見を検討することを目的とした。両者とも2枚のβ-シートの間にはさまれたクレフトと考えられる領域に活性部位が存在し触媒活性を直接担っていると考えられている。両酵素のα,β領域とβ,β領域を連結するヒンジ部分でそれぞれの領域が分断できる構造のため、両酵素遺伝子のこれらヒンジ部分近傍の塩基配列に部位特異的変異操作により新たに制限酵素部位を付加し、両遺伝子の各領域に相当する間を合成リンカーで連結するか、または制限酵素部位を付加する場合が不可能な場合はPCRによって増幅した各遺伝子のDAN断片を連結することで2種類のキメラ遺伝子[XCX,CXCと仮称]を構築した。それらの遺伝子構造は塩基配列から確認した。さらに大腸菌発現ベクターに挿入した両キメラ遺伝子を保持する各形質転換株の全蛋白質をSDS-PAGEで分析したところ、予想された分子量に相当する蛋白質バンドの存在が確認された。さらに、これら蛋白質は両酵素に対する抗血清と反応することがウェスタンブロッティングにより確認されたことから、両キメラ蛋白質の発現していることが確認された。
  • 文部科学省:科学研究費補助金(奨励研究(A))
    研究期間 : 1993年 -1993年 
    代表者 : 大井 俊彦
     
    Aspergillus aculeatus由来のセルラーゼFI-CMCaseをコードするcDNAがクローン化され、大腸菌における高発現系も構築した。さらにX線結晶解析により立体構造も解明され、本酵素分子は2枚のb-シートに挟まれたクレフトと考えられる部分に触媒部位が存在すると考えられる。部位特異的変異操作の結果から、このクレフト内に存在するGlu118-Glu202のアミノ酸残基のペアが直接触媒活性を担っているものと考えられるが、Glu118近傍に存在するAsp101が触媒活性に大きく影響していることが明らかとなった。そこでAsp101に影響されてGlu118の解離が促進されGlu202との間にpk値の勾配を作ることで触媒機能を有する3つのアミノ酸残基の共同作用による触媒機構を想定し、Aps101の触媒活性における役割を解明するために計画した。Fl-CMCase遺伝子のAsp101に相当するアミノ酸コドンを以下のように部位特異的変異操作によってSer(D101S)およびGlu(D101E)にそれぞれ改変し、大腸菌において大量に発原させた変異酵素を精製してこれら異変酵素の酵素活性を反応速度論的に解析することでAsp101およびGlu118の触媒機構における役割を考察した。作製した2種の変異酵素D101EおよびD101Sを野性型酵素とともに反応速度論的に比較解析したところ、km値には両変異酵素とも大きな変化が見られなかったが、Vm値は野性型酵素に比べて大きく変化し、D101Eでは約1/(1000)に、D101Sにおいては約1/(10000)にそれぞれ低下した。このことはAsp101はFl-CMCaseの触媒活性に重大な影響を与えており、本酵素の立体構造から考えられる触媒残基の一つであるGlu118の持っている本来のpk値になんらかの影響を与えたことで、著しい酵素活性の低下が起きたことを示唆している。

産業財産権



Copyright © MEDIA FUSION Co.,Ltd. All rights reserved.