2021/04/02 更新

写真a

イワサキ ユウゴ
岩崎 雄吾
IWASAKI, Yugo
所属
大学院生命農学研究科 応用生命科学専攻 応用生命科学 准教授
大学院担当
大学院生命農学研究科
学部担当
農学部
職名
准教授
連絡先
メールアドレス
外部リンク

学位 2

  1. 農学博士 ( 名古屋大学 ) 

  2. 農学修士 ( 名古屋大学 ) 

研究キーワード 7

  1. 脂質の合成

  2. 脂質の分析

  3. 酵素工学

  4. 酵素の改変

  5. 蛋白質工学

  6. 脂質工学

  7. 脂質の分析

研究分野 2

  1. ライフサイエンス / 応用微生物学

  2. 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

現在の研究課題とSDGs 2

  1. リン脂質類の高純度精密合成

  2. 微生物ホスリパーゼに関する研究

経歴 4

  1. 名古屋大学大学院生命農学研究科   准教授

    2007年4月

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    国名:日本国

  2. 名古屋大学大学院生命農学研究科   助教授

    2005年4月 - 2007年3月

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    国名:日本国

  3. 名古屋大学   講師

    2001年8月 - 2005年3月

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    国名:日本国

  4. 名古屋大学   助手

    1995年7月 - 2001年7月

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    国名:日本国

学歴 3

  1. 名古屋大学   大学院生命農学研究科 博士(農学)

    1998年6月

  2. 名古屋大学   農学研究科   食品工業化学

    1991年4月 - 1993年3月

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    国名: 日本国

  3. 名古屋大学   農学部   食品工業化学

    1987年4月 - 1991年3月

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    国名: 日本国

所属学協会 5

  1. 日本生物工学会

  2. 日本農芸化学会

  3. 日本油化学会

  4. 酵素工学研究会

  5. バイオインダストリー協会

委員歴 3

  1. 日本農芸化学会   産学官学術交流委員  

    2018年 - 現在   

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    団体区分:学協会

  2. Journal of Bioscience and Biotechnology編集委員会   編集委員  

    2011年6月 - 2015年5月   

  3. 日本油化学会東海支部   常任幹事  

    1995年 - 現在   

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    団体区分:学協会

受賞 3

  1. 第10回酵素応用シンポジウム研究奨励賞

    2009年6月   天野エンザイム  

    岩崎 雄吾

  2. 日本油脂工業会館優秀技術論文賞

    2000年2月   日本油脂工業会館  

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    受賞国:日本国

  3. バイオインダストリー協会「発酵と代謝」研究奨励金受領者

    1999年11月   日本バイオインダストリー協会  

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    受賞国:日本国

 

論文 80

  1. 改変型ホスホリパーゼDによる難合成性リン脂質の合成 招待有り

    岩崎雄吾, Jasmina Damnjanović

    生物工学   98 巻 ( 9 ) 頁: 481 - 484   2020年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

  2. A simple chemo-enzymatic synthesis of alkyl-acyl (plasmanyl) phospholipids 招待有り 査読有り

    Yugo Iwasaki, Yuuki Sakurai, Jasmina Damnjanović

    Biocatalysis and Agricultural Biotechnology   26 巻   頁: 101625 - 101625   2020年7月

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    担当区分:筆頭著者, 責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Elsevier BV  

    DOI: 10.1016/j.bcab.2020.101625

  3. Acyl chain that matters: Introducing sn-2 acyl chain preference to a phospholipase D by protein engineering. 査読有り

    Jasmina Damnjanović, Hideo Nakano, Yugo Iwasaki

    Protein Eng. Des. Sel.   32 巻   頁: 1 - 11   2019年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1093/protein/gzz019

  4. Production of active Manganese peroxidase in Escherichia coli by co-expression of chaperones and in vitro maturation by ATP-dependent chaperone release. 査読有り

    Alfi A, Zhu, Bou, Jasmina Damnjanović, Takaaki Kojima, Yugo Iwasaki, Hideo Nakano

    J. Biosci. Bioeng.   128 巻   頁: 290 - 295   2019年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1016/j.jbiosc.2019.02.011

  5. 生物工学分野における分子ドッキングシミュレーションの応用 招待有り

    ダムニャノビッチ ヤスミナ, 岩崎雄吾

    生物工学会誌   97 巻 ( 5 ) 頁: 257 - 260   2019年

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    記述言語:日本語   掲載種別:研究論文(学術雑誌)  

  6. Scrambling of natural and fluorescently tagged phosphatidylinositol by reconstituted G protein-coupled receptor and TMEM16 scramblases. 査読有り 国際共著

    Wang L, Iwasaki Y, Andra KK, Pandey K, Menon AK, Bütikofer P

    The Journal of Biological Chemistry     頁: 18318 - 18327   2018年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1074/jbc.RA118.004213

    PubMed

  7. Enzymatic modification of phospholipids 招待有り

    Damnjanović, J, Iwasaki, Y

    “Recent Advances in Biotechnology Vol. 4. Progress in Food Biotechnology” (ed. by Ali Osman).     頁: 82 - 131   2018年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

  8. Facile Enzymatic Synthesis of Phosphatidylthreonine Using an Engineered Phospholipase D

    Jasmina Damnjanović, Nozomi Matsunaga, Masaatsu Adachi, Hideo Nakano, Yugo Iwasaki

    European Journal of Lipid Science and Technology   120 巻 ( 6 )   2018年6月

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    担当区分:責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Wiley-VCH Verlag  

    Here is described the newly established method for direct enzymatic synthesis of phosphatidylthreonine (PtdThr). It is the first report on enzymatic synthesis of this rare phospholipid. It utilizes phospholipase D (PLD)-catalyzed transphosphatidylation, in which the head group of phosphatidylcholine (PtdCho) is exchanged to L-Threonine (L-Thr). An attempt to catalyze the reaction between PtdCho and L-Thr using wild-type PLD is not successful, possibly because the secondary hydroxyl group of L-Thr is not accessible to the enzyme. To synthesize Ptd-L-Thr, the natural PtdThr isomer, engineered PLD variants active toward secondary hydroxyls of inositol are screened for their ability to accept L-Thr. Six variants are identified as positive, among which 187F/191Y/385L (FYL) shows highest activity. After optimizing the reaction parameters, Ptd-L-Thr content reaches approximately 30 mol%. The product is isolated by column chromatography with the overall yield of 5.2%, and its structure is confirmed by NMR. In addition, the FYL variant can also react on some stereoisomers of threonine, L-allo-Thr, and D-allo-Thr as well as L-Thr, but not D-Thr. Ligand docking simulation explains the enzyme's preference toward these stereoisomers
    L-, L-allo-, and D-allo-Thr can bind to the enzyme's active site in a productive orientation, whereas D-Thr binds in a position which makes the reaction impossible to proceed. Practical Applications: The enzymatic method enables one-step synthesis of PtdThr from PtdCho and L-Threonine without any protection/deprotection steps, thereby being much more simple and less hazardous than the currently used chemical methods. The synthesized PtdThr can be isolated in pure form and used as a reagent for elucidation of its biological functions. A method for one-step enzymatic synthesis of phosphatidylthreonine (PtdThr) is described. It uses the head group exchange of phosphatidylcholine (PtdCho) with Threonine(Thr) catalyzed by an engineered phospholipase D (PLD). The enzyme can accept some stereoisomers of Thr, that is, L-Thr, L-allo-Thr, and D-allo-Thras the substrates to give the corresponding PtdThr, but notD-Thr. Docking simulation explainsthe enzyme's preference toward thesestereoisomers
    L-, L-allo-, and D-allo-Thr can bind to the enzyme's active site in a productive orientation, whereas D-Thrbinds in a non-productive orientation which makes the reaction impossible to proceed.

    DOI: 10.1002/ejlt.201800089

    Web of Science

    Scopus

  9. Directing positional specificity in enzymatic synthesis of bioactive 1-phosphatidylinositol by protein engineering of a phospholipase D 査読有り

    Jasmina Damnjanovic, Chisato Kuroiwa, Hidetoshi Tanaka, Ken Ishida, Hideo Nakano, Yugo Iwasaki

    BIOTECHNOLOGY AND BIOENGINEERING   113 巻 ( 1 ) 頁: 62 - 71   2016年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY-BLACKWELL  

    Phosphatidylinositol (PI) holds a potential of becoming an important dietary supplement due to its effects on lipid metabolism in animals and humans manifested as a decrease of the blood cholesterol and lipids, and relief of the metabolic syndrome. To establish an efficient, enzymatic system for PI production from phosphatidylcholine and myo-inositol as an alcohol acceptor, our previous study started with the wild-type Streptomyces antibioticus phospholipase D (SaPLD) as a template for generation of PI-synthesizing variants by saturation mutagenesis targeting positions involved in acceptor accommodation, W187, Y191, and Y385. The isolated variants generated PI as a mixture of positional isomers, among which only 1-PI exists in nature. Thus, the current study has focused to improve positional specificity of W187N/Y191Y/Y385R SaPLD (NYR) which generates PI as a mixture of 1-PI and 3-PI in the ratio of 76/24, by subjecting four residues of its acceptor-binding site to saturation mutagenesis. Subsequent screening pointed at NYR-186T and NYR-186L as the most improved variants producing PI with a ratio of 1-/3-PI=93/7 and 87/13, respectively, at 37 degrees C. Lowering the reaction temperature further improved the specificity of both variants to 1-/3-PI>97/3 at 20 degrees C with no change in total PI yield. Structure model analyses imply that G186T and G186L mutations increased rigidity of the acceptor-binding site, thus limiting the possible orientations of myo-inositol. The two newly isolated PLDs are promising for future application in large-scale 1-PI production. Biotechnol. Bioeng. 2016;113: 62-71. (c) 2015 Wiley Periodicals, Inc.

    DOI: 10.1002/bit.25697

    Web of Science

    PubMed

  10. Extracellular production of Pseudozyma (Candida) antarctica lipase B with genuine primary sequence in recombinant Escherichia coli. 査読有り

    Ayana Ujiie, Hideo Nakano and Yugo Iwasaki

    J. Biosci. Bioeng.   121 巻   頁: 303–309   2016年

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    担当区分:筆頭著者   記述言語:英語   掲載種別:研究論文(学術雑誌)  

  11. Signal peptide optimization tool for the secretion of recombinant protein from Saccharomyces cerevisiae 査読有り

    Akihiro Mori, Shoichi Hara, Tomohiro Sugahara, Takaaki Kojima, Yugo Iwasaki, Yasuaki Kawarasaki, Takehiko Sahara, Satoru Ohgiya, Hideo Nakano

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   120 巻 ( 5 ) 頁: 518 - 525   2015年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SOC BIOSCIENCE BIOENGINEERING JAPAN  

    The secretion efficiency of foreign proteins in recombinant microbes is strongly dependent on the combination of the signal peptides (SPs) used and the target proteins; therefore, identifying the optimal SP sequence for each target protein is a crucial step in maximizing the efficiency of protein secretion in both prokaryotes and eukaryotes. In this study, we developed a novel method, named the SP optimization tool (SPOT), for the generation and rapid screening of a library of SP-target gene fusion constructs to identify the optimal SP for maximizing target protein secretion. In contrast to libraries generated in previous studies, SPOT fusion constructs are generated without adding the intervening sequences associated with restriction enzyme digestion sites. Therefore, no extra amino acids are inserted at the N-terminus of the target protein that might affect its function or conformational stability. As a model system, beta-galactosidase (LacA) from Aspergillus oryzae was used as a target protein for secretion from Saccharomyces cerevisiae. In total, 60 SPs were selected from S. cerevisiae secretory proteins and utilized to generate the SP library. While many of the SP-LacA fusions were not secreted, several of the SPs, AGA2, CRH1, PLB1, and MF(alpha)1, were found to enhance LacA secretion compared to the WT sequence. Our results indicate that SPOT is a valuable method for optimizing the bioproduction of any target protein, and could be adapted to many host strains. (C) 2015, The Society for Biotechnology, Japan. All rights reserved.

    DOI: 10.1016/j.jbiosc.2015.03.003

    Web of Science

    PubMed

  12. Role of disulfide bond isomerase DsbC, calcium ions, and hemin in cell-free protein synthesis of active manganese peroxidase isolated from Phanerochaete chrysosporium 査読有り

    Ryoko Ninomiya, Bo Zhu, Takaaki Kojima, Yugo Iwasaki, Hideo Nakano

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   117 巻 ( 5 ) 頁: 652 - 657   2014年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SOC BIOSCIENCE BIOENGINEERING JAPAN  

    A cell-free protein synthesis system can produce various types of proteins directly from DNA templates such as PCR products, and therefore attracts great attention as an alternative protein synthesis system especially for high-throughput functional screening of proteins. Here, we report successful expression of active Phanerochaete chrysosporium manganese peroxidase (MnP) in an Escherichia colt cell-free protein synthesis system, wherein reaction conditions such as the concentrations of hemin, calcium ions, and disulfide bond isomerase were optimized to increase the solubility and activity of the synthesized enzyme. Moreover, cell-free synthesized MnP purified using the hemagglutinin tag showed higher specific activity than the commercial wild-type enzyme, suggesting that the cell-free system can be used as a preparative method for efficient synthesis of disulfide bond-containing metalloenzymes such as MnP. We believe that our system is a solid foundation for the development of a high-throughput screening method for the directed evolution of these enzymes. (C) 2013, The Society for Biotechnology, Japan. All rights reserved.

    DOI: 10.1016/j.jbiosc.2013.11.003

    Web of Science

    PubMed

  13. Deletion of a Dynamic Surface Loop Improves Stability and Changes Kinetic Behavior of Phosphatidylinositol-Synthesizing Streptomyces Phospholipase D 査読有り

    Jasmina Damnjanovic, Hideo Nakano, Yugo Iwasaki

    BIOTECHNOLOGY AND BIOENGINEERING   111 巻 ( 4 ) 頁: 674 - 682   2014年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY-BLACKWELL  

    Supplementary phosphatidylinositol (PI) was shown to improve lipid metabolism in animals, thus it is interesting for pharmaceutical and nutritional applications. Homogenous PI can be produced in transphosphatidylation of phosphatidylcholine (PC) with myo-inositol catalyzed by phospholipase D (PLD). Only bacterial enzymes able to catalyze PI synthesis are Streptomyces antibioticus PLD (SaPLD) variants, among which DYR (W187D/Y191Y/Y385R) has the best kinetic profile. Increase in PI yield is possible by providing excess of solvated myo-inositol, which is achievable at high temperatures due to its highly temperature-dependent solubility. However, high-temperature PI synthesis requires the thermostable PLD. Previous site-directed combinatorial mutagenesis at the residues of DYR having high B-factor yielded the most improved variant, D40H/T291Y DYR, obtained by the combination of two selected mutations. D40 and T291 are located within dynamic surface loops, D37-G45 (termed D40 loop) and G273-T313. Thus, in this work, thermostabilization of DYR SaPLD was attempted by rational design based on deletion of the D40 loop, generating two variants, 37-45 DYR and 38-46 DYR PLD. 38-46 DYR showed highest thermostability as its activity half-life at 70 degrees C proved 11.7 and 8.0 times longer than that of the DYR and 37-45 DYR, respectively. Studies on molecular dynamics predicted 38-46 DYR to have the least average RMSD change as temperature dramatically increases. At 60 and 70 degrees C, both mutants synthesized PI in a twofold higher yield compared to the DYR, while at the same time produced less of the hydrolytic side-product, phosphatidic acid. Biotechnol. Bioeng. 2014;111: 674-682. (c) 2013 Wiley Periodicals, Inc.

    DOI: 10.1002/bit.25149

    Web of Science

    PubMed

  14. A chromogenic substrate for solid-phase detection of phospholipase A₂. 査読有り

    Eba C, Okano A, Nakano H, Iwasaki Y

    Analytical biochemistry   447 巻   頁: 43 - 45   2014年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1016/j.ab.2013.11.007

    Web of Science

    PubMed

  15. Phospholipase D as a catalyst: Application in phospholipid synthesis, molecular structure and protein engineering 査読有り

    Jasmina Damnjanovic, Yugo Iwasaki

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   116 巻 ( 3 ) 頁: 271 - 280   2013年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SOC BIOSCIENCE BIOENGINEERING JAPAN  

    Phospholipase D (PLD) is a useful enzyme for its transphosphatidylation activity, which enables the enzymatic synthesis of various phospholipids (PLs). Many reports exist on PLO-mediated synthesis of natural and tailor-made PLs with functional head groups, from easily available lecithin or phosphatidylcholine. Early studies on PLO-mediated synthesis mainly employed enzymes of plant origin, which were later supplanted by ones from microorganisms, especially actinomycetes. Many PLDs are members of the PLD superfamily, having one or two copies of a signature sequence, HxKxxxxD or HKD motif, in the primary structures. PLO superfamily members share a common core structure, and thereby, a common catalytic mechanism. The catalysis proceeds via two-step reaction with the formation of phosphatidyl-enzyme intermediate. Both of the two catalytic His residues are critical in the reaction course, where one acts as a nucleophile, while the other functions as a general acid/base. PLD is being engineered to improve its activity and stability, alter head group specificity and further identify catalytically important residues. Since the knowledge on PLD enzymology is constantly expanding, this review focuses on recent advances in the field, regarding PLD-catalyzed synthesis of bioactive PLs, deeper understanding of substrate recognition and binding mechanism, altering substrate specificity, and improving thermostability. We introduced some of our recent results in combination with existing facts to further deepen the story on the nature of this useful enzyme. (C) 2013, The Society for Biotechnology, Japan. All rights reserved.

    DOI: 10.1016/j.jbiosc.2013.03.008

    Web of Science

    PubMed

  16. Simple and Efficient Profiling of Phospholipids in Phospholipase D-modified Soy Lecithin by HPLC with Charged Aerosol Detection 査読有り

    Jasmina Damnjanovic, Hideo Nakano, Yugo Iwasaki

    JOURNAL OF THE AMERICAN OIL CHEMISTS SOCIETY   90 巻 ( 7 ) 頁: 951 - 957   2013年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SPRINGER  

    Dietary phosphatidylinositol (PI) can be synthesized via phospholipase D (PLD)-catalyzed transphosphatidylation of phosphatidylcholine (PC), abundant in soy lecithin, with myo-inositol. However, a generated mixture of phospholipid (PL) classes poses a challenge for analysis. Our current work on Streptomyces PLD engineering requires a robust analytical method for profiling of PI and related PLs derived from the transphosphatidylation reactions. Therefore, we optimized an HPLC-based method with charged aerosol detector (CAD) for PL quantification. PLs were separated on a normal phase silica column by a gradient elution system using two solvents containing chloroform/methanol/1 M formic acid-triethylamine buffer in different ratios. Retention times of the PL standards and LC-MS under identical conditions were used to identity PL classes. PL standards gave linear response in 100- and 10-fold (lyso-PI) concentration range. The method provided a simple, sensitive, repeatable, and precise analysis of PI, PC, phosphatidylethanolamine, phosphatidic acid, and lyso forms of PC and PI. Compared to the similar existing method, introduction of CAD provided a three- to fivefold decrease at the lower end and a two- to fivefold increase at the upper end of the dynamic range. High precision, high sensitivity, and low limits of detection and quantification further underline the benefits of CAD in PL analysis.

    DOI: 10.1007/s11746-013-2236-x

    Web of Science

  17. 放線菌ホスホリパーゼDの改変とリン脂質合成への応用 招待有り 査読有り

    岩崎雄吾

    オレオサイエンス   13 巻   頁: 465-469   2013年

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    担当区分:筆頭著者   記述言語:日本語   掲載種別:研究論文(学術雑誌)  

  18. Investigation of β-Cryptoxanthin Fatty Acid Ester Compositions in Citrus Fruits Cultivated in Japan. 査読有り

    Wada, Y., Matsubara, A., Uchikata, T., Iwasaki, Y., Morimoto, S., Kan, K., Ookura, T., Fukusaki, E., and Bamba, T.

    Food Nutr. Sci.   4 巻   頁: 98-104   2013年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

  19. Improving thermostability of phosphatidylinositol-synthesizing Streptomyces phospholipase D 査読有り

    Jasmina Damnjanovic, Rie Takahashi, Atsuo Suzuki, Hideo Nakano, Yugo Iwasaki

    PROTEIN ENGINEERING DESIGN & SELECTION   25 巻 ( 8 ) 頁: 415 - 424   2012年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:OXFORD UNIV PRESS  

    Aimed to produce thermostable phosphatidylinositol (PI)-synthesizing phospholipase D (PLD), we initiated site-directed combinatorial mutagenesis followed by high-throughput screening. Previous site-directed combinatorial mutagenesis of wild-type Streptomyces PLD produced a mutant, DYR (W187D/Y191Y/Y385R) with PI-synthesizing ability. Deriving PI as a product of transphosphatidylation between phosphatidylcholine and myo-inositol, with myo-inositol in excess at high-temperature reaction conditions can increase yield due to enhanced solubility of this substrate. Thus, we improved DYRs thermostability by introduction of random mutations into selected amino acid positions having high B-factor. Screening of the libraries under restricted conditions yielded single-point mutants, specifically D40H, T291Y and R329G. Combinations of these point mutations yielded double (D40H/T291Y, D40H/R329G and T291Y/R329G) and triple (D40H/T291Y/R329G) mutants. PI synthesis at elevated temperatures pointed at D40H/T291Y as the most efficient enzyme. Circular dichroism analysis revealed D40H/T291Y to have increased melting temperature and postponed onset of thermal unfolding compared with DYR. Thermal tolerance study at 65C confirmed D40H/T291Ys thermostability as its half-inactivation time was 8.7 min longer compared with DYR. This mutant had significantly less root-mean-square deviation change compared with DYR and showed no change in root-mean-square fluctuation when temperature shifts from 40 to 60C, as determined by molecular dynamics analysis. Acquired different degrees of thermostability were also observed for several other DYR mutants.

    DOI: 10.1093/protein/gzs038

    Web of Science

    PubMed

  20. Metabolic profiling of ss-cryptoxanthin and its fatty acid esters by supercritical fluid chromatography coupled with triple quadrupole mass spectrometry 査読有り

    Yusuke Wada, Atsuki Matsubara, Takato Uchikata, Yugo Iwasaki, Satoshi Morimoto, Katsuta Kan, Tetsuya Okura, Eiichiro Fukusaki, Takeshi Bamba

    JOURNAL OF SEPARATION SCIENCE   34 巻 ( 24 ) 頁: 3546 - 3552   2011年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY PERIODICALS, INC  

    In this study, a high-throughput and high-sensitivity profiling system for beta-cryptoxanthin (beta CX) and beta-cryptoxanthin fatty acid ester (beta CXFA) was constructed by supercritical fluid chromatography (SFC) coupled with triple quadrupole mass spectrometry (QqQMS). beta CX and nine beta CXFAs were successfully separated within 20 min using a column packed with octadecylsilyl-bonded silica particles. The limit of detection was 540 fmol for the free form and 32130 fmol for the esterified forms. These results demonstrate that both the throughput and the sensitivity of this SFC-QqQMS system are considerably higher than those of conventional methods. When this system was applied for the analysis of Citrus unshiu, beta CX and five beta CXFAs were directly detected with much simpler sample pre-preparation. The analysis of other citrus fruits indicated that the beta CXFA profiles varied with their breed variety. Furthermore, gas chromatography-mass spectrometry was used to analyze total fatty acid profiles in C. unshiu, and the results revealed that the profiles of fatty acids located in beta CXFA were distinct. This is the first report on the analysis of beta CX and its fatty acid derivatives by SFC-QqQMS. The profiling system developed in this study will be a powerful tool for investigating xanthophyll fatty acid esters.

    DOI: 10.1002/jssc.201100376

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  21. Synthesis of Phospholipids Containing Polyunsaturated Fatty Acids by Phospholipase A(2)-mediated Esterification with Food-compatible Reagents 査読有り

    Tatsushi Tanaka, Tetsuo Isezaki, Hideo Nakano, Yugo Iwasaki

    JOURNAL OF OLEO SCIENCE   59 巻 ( 7 ) 頁: 375 - 380   2010年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:JAPAN OIL CHEMISTS SOC  

    Enzymatic synthesis of phospholipids (PLs) containing polyunsaturated fatty acids (PUFAs) was studied. The main purpose was to establish an efficient production method for PLs containing docosahexaenoic acid or eicosapentaenoic acid using only food-compatible reagents. Phospholipase A(2) (PLA(2))-mediated ester synthesis was employed to introduce the PUFAs into the sn-2 position of lysophospholipid (LPL) to yield PUFA-containing PLs. When LPL and the fatty acids were reacted in glycerol in the presence of porcine pancreas PLA(2), the reaction was not very effective. However, it was found that addition of certain kinds of amino acids such as glycine or L-alanine in the reaction mixture improved the reaction. After the reaction, the synthesized PLs were extracted selectively with ethanol and n-hexane, leaving the unreacted LPL, amino acids and the enzyme remained in the glycerol layer. It was confirmed that the enzyme remained in the glycerol layer could be reused by adding fresh substrates for the subsequent reactions.

    DOI: 10.5650/jos.59.375

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  22. Synthesis of phosphatidylinositols having various inositol stereoisomers by engineered phospholipase D 査読有り

    Akari Ozaki, Atsushi Masayama, Hideo Nakano, Yugo Iwasaki

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   109 巻 ( 4 ) 頁: 337 - 340   2010年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SOC BIOSCIENCE BIOENGINEERING JAPAN  

    Phospholipase D-mediated synthesis of phosphatidylinositols having various inositol stereoisomers was studied. Seven inositol stereoisomers were tested, all of which were found to be substrates of the enzyme, generating the corresponding phosphatidylinositols. Based on the substrate specificity, models for the recognition of inositol by the enzyme were proposed. (C) 2009, The Society for Biotechnology, Japan. All rights reserved.

    DOI: 10.1016/j.jbiosc.2009.09.045

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  23. Composition analysis of positional isomers of phosphatidylinositol by high-performance liquid chromatography 査読有り

    Yugo Iwasaki, Atsushi Masayama, Akihiro Mori, Chika Ikeda, Hideo Nakano

    JOURNAL OF CHROMATOGRAPHY A   1216 巻 ( 32 ) 頁: 6077 - 6080   2009年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER SCIENCE BV  

    An HPLC-based method has been developed for composition analysis of six positional isomers of phosphatidylinositol (PI), of which the phosphatidyl group was connected to different positions of the myo-inositol moiety. The method employed a combination of two types of HPLC analyses. One was direct separation of the six PI isomers into four peaks of 1(3)-PI, 2-PI, 4(6)-PI and 5-PI on a normal-phase silica gel column. The second method was for the separations of I -PI from 3-PI and 4-PI from 6-PI, which were not separable on the normal-phase column. This method involved conversion of PI isomers into pentakis-(R)-1-phenylethylcarbamate (PEC) derivatives, which were separated on a reversed-phase column. Using the established method, positional specificity of several engineered phospholipases D in enzymatic synthesis of PI from myo-inositol and phosphatidylcholine was investigated. This was performed by analyzing the isomeric composition of PIs synthesized by the mutant enzymes. Among five mutant enzymes tested, two showed strong specificity to 1-OH, one showed moderate preference to 1-OH, one preferred 3-OH, and one showed broad specificity towards 1-, 3-,4- and 6-OH. (C) 2009 Elsevier B.V. All rights reserved.

    DOI: 10.1016/j.chroma.2009.06.064

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  24. Enzymatic preparation of enantiomerically pure sn-2,3-diacylglycerols: A stereoselective ethanolysis approach

    Weera Piyatheerawong, Tsuneo Yamane, Hideo Nakano, Yugo Iwasaki

    JOURNAL OF THE AMERICAN OIL CHEMISTS SOCIETY   83 巻 ( 7 ) 頁: 603 - 607   2006年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER OIL CHEMISTS SOC A O C S PRESS  

    Stereoselective ethanolysis of monoacid TAG by immobilized Rhizomucor miehei lipase (RML) was studied for preparation of optically pure sn-2,3-DAG. Trioctanoylglycerol (TO) was used as a model substrate. The enantiomeric purity of the product, sn-2,3-dioctanoylglycerol (sn-2,3-DO), was very high (percent enantiomeric excess > 99%) when an excess of ethanol was used. The result indicated that RML was highly stereoselective toward the sn-1 position of TO under conditions of excess ethanol. The stereoselectivity of RML depended on the amount of ethanol. The larger the amount of ethanol was, the higher the stereoselectivity, became. After optimizing the parameters such as reactant molar ratio, water content, and temperature, (ethanol/TO molar ratio = 31:1 and water content = 7.5 wt% of the reactants at 25 degrees C), optically pure sn-2,3-DO was obtained at 61.1 mol% in the glyceride fraction in 20 min. The above conditions were further applied for ethanolysis of monoacid TAG with different acyl groups such as triclecanoylglycerol (C10:0), tridoclecanoylglycerol (C12:0), tritetradecanoylglycerol (C14:0) and trioctadecenoylglycerol [triolein, (C18:1)]. The yields and enantiomeric purities of 1,2(2,3)-DAG were dramatically reduced when TAG with FA longer than decanoic acid were used.

    DOI: 10.1007/s11746-006-1245-4

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  25. Direct separation of regio- and enantiomeric isomers of diacylglycerols by a tandem column high-performance liquid chromatography 査読有り

    W Piyatheerawong, Y Iwasaki, T Yamane

    JOURNAL OF CHROMATOGRAPHY A   1068 巻 ( 2 ) 頁: 243 - 248   2005年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER SCIENCE BV  

    A novel HPLC-based method for direct separation of the three isomers of mono-acid diacylglycerols (DAGs), i.e., 1,2-DAG, 2,3-DAG and 1,3-DAG. has been established. The method employs a tandem column system, in which two different columns (a conventional silica gel column and a chiral stationary phase column) are connected in series. Two isomeric mixtures of DAGs (i.e., dicapryloylglycerol and dioleoylalycerol) and lipase-catalyzed reaction mixtures were successfully resolved on the tandem column HPLC system without any derivatization prior to the analysis. According to the established analytical method, stereoselectivity of two lipases toward mono-acid triacylglycerols in ethanolysis reaction was investigated. The tested enzymes were immobilized Candida antarctica lipase B (CALB) and Rhizomucor miehei lipase (RML). Analyses of the enantiomeric purity of 1,2-DAG and 2,3-DAG, generated as intermediates during the reaction, revealed that CALB and RML have sn-3 and sn-1 stereopreference, respectively. (c) 2005 Elsevier B.V. All rights reserved.

    DOI: 10.1016/j.chroma.2005.01.075

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  26. Detection of phospholipase D on solid materials

    Y Iwasaki, S Nishikawa, M Tsuneda, T Takahashi, T Yamane

    ANALYTICAL BIOCHEMISTRY   329 巻 ( 1 ) 頁: 157 - 159   2004年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ACADEMIC PRESS INC ELSEVIER SCIENCE  

    DOI: 10.1016/j.ab.2004.02.040

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  27. Dependency of water concentration on ethanolysis of trioleoylglycerol by lipases

    W Piyatheerawong, Y Iwasaki, XB Xu, T Yamane

    JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC   28 巻 ( 1 ) 頁: 19 - 24   2004年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER SCIENCE BV  

    The effects of water concentration on ethanolysis of trioleoylglycerol catalyzed by four different lipases were studied. The target product of the ethanolysis was 2-monooleoylglycerol (2-MO). Novozym 435 (a commercially available preparation of immobilized Candida antarctica lipase B, CALB) exhibited both the highest product yield and the reaction rate at very low (less than 1 wt.%) free water concentration. Its catalytic activity did not drop even in dry state, i.e. in the system of dry CALB in dry ethanol (water concentration was ca. 0.1 wt.%). In contrast, other three immobilized lipases tested (Rhizomucor miehei lipase, Burkholderia cepacia lipase and Thermomyces lanuginosus lipase) required larger amounts of free water (ca. 7-9 wt.%) for their best performance and exhibited no ethanolysis reaction at low free water concentrations. The CALB's anomalous behavior was also observed in other two different preparations of CALB; i.e., free CALB powder and silica-immobilized CALB as well as Novozym 435. Thus, it was confirmed that no extra water requirement of CALB was an intrinsic property of CALB itself. No extra water requirement of CALB implies that this enzyme is able to keep water molecules tightly to retain its catalytic activity even in dry ethanol (a water-depriving solvent). It is suggested that some structural features, such as a carbohydrate molecule, a lid-like domain, or very tight bonding of the essential water molecule(s), might be involved in this very unique property. (C) 2004 Elsevier B.V. All rights reserved.

    DOI: 10.1016/j.moleatb.2004.01.008

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  28. Enzymatic synthesis of structured lipids

    Y Iwasaki, T Yamane

    RECENT PROGRESS OF BIOCHEMICAL AND BIOMEDICAL ENGINEERING IN JAPAN I   90 巻   頁: 151 - 171   2004年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SPRINGER-VERLAG BERLIN  

    Structured lipids (SLs) are defined as lipids that are modified chemically or enzymatically in order to change their structure. This review deals with structured triacylglycerols (STGs) and structured phospholipids (SPLs). The most typical STGs are MLM-type STGs, having medium chain fatty acids (FAs) at the 1- and 3-positions and a long chain fatty acid at the 2-position. MLM-type STGs are synthesized by: 1) 1,3-position-specific lipase-catalyzed acyl exchange of TG with FA or with FA ethylester (FAEt); 2) 1,3-position-specific lipase-catalyzed acylation of glycerol with FA, giving symmetric 1,3-diacyl-sn-glycerol, followed by chemical acylation at the sn-2 position, and; 3) 1,3-position-specific lipase-catalyzed deacylation of TG, giving 2-monoacylglycerol, followed by reacylation at the 1- and 3-positions with FA or with (FAEt). Enzymatic preparation of SPLs requires: 1) acyl group modification, and 2) head group modification of phospholipids. Acyl group modification is performed using lipases or phospholipase A(2)-mediated transesterification or ester synthesis to introduce arbitrary fatty acid to phospholipids. Head group modification is carried out by phospholipase D-catalyzed transphosphatidylation. A wide range of compounds can be introduced into the polar head of phospholipids, making it possible to prepare various SPLs.

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  29. ホスホリパーゼDの酵素工学 招待有り

    岩崎雄吾, 山根恒夫

    生物工学会誌   82 巻   頁: 284 - 299   2004年

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    記述言語:日本語   掲載種別:研究論文(学術雑誌)  

  30. An aqueous suspension system for phospholipase D-mediated synthesis of PS without toxic organic solvent

    Y Iwasaki, Y Mizumoto, T Okada, T Yamamoto, K Tsutsumi, T Yamane

    JOURNAL OF THE AMERICAN OIL CHEMISTS SOCIETY   80 巻 ( 7 ) 頁: 653 - 657   2003年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER OIL CHEMISTS SOC A O C S PRESS  

    Enzymatic synthesis of PS by phospholipase D (PLD)-mediated transphosphatidylation in an aqueous media was investigated. The purpose of this study was to establish a novel synthetic method where no toxic organic solvents were used. An attempt to react soybean lecithin (simply dispersed in an aqueous buffer) with an aqueous solution Of L-serine and PLD was unsuccessful, giving only 20% of PS. By contrast, a suspension of lecithin adsorbed on fine powders such as silica was effectively converted into PS in an aqueous solution Of L-serine and PLD. After screening various powders for use as the lecithin adsorbent, calcium sulfate was found to be the best with respect to lecithin conversion. In addition, calcium sulfate did not require prior adsorption of lecithin (i.e., the reaction proceeded effectively simply by adding the powder to an aqueous mixture of lecithin, L-serine, and PLD). With this "aqueous suspension system" of calcium sulfate, up to 180 mg/mL lecithin was completely converted, resulting in more than 80% PS in 24 h. The synthesized PS could easily be recovered from the powder by extracting with a mixture of n-hexane, ethanol, and diluted HCl.

    DOI: 10.1007/s11746-003-0754-5

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  31. Crystal structure of bovine trypsin and wheat germ trypsin inhibitor (I-2b) complex at 2.3 A° resolution 査読有り

    Raj SSS, Kibushi E, Kurasawa T, Suzuki A, Yamane T, Odani S, Iwasaki Y, Yamane T, Ashida T

    J. Biochem.   132 巻 ( 6 ) 頁: 927 - 933   2002年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  32. Study of TAG ethanolysis to 2-MAG by immobilized Candida antarctica lipase and synthesis of symmetrically structured TAG

    R Irimescu, Y Iwasaki, CT Hou

    JOURNAL OF THE AMERICAN OIL CHEMISTS SOCIETY   79 巻 ( 9 ) 頁: 879 - 883   2002年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER OIL CHEMISTS SOC A O C S PRESS  

    Regiospecific ethanolysis of homogenous TAG with immobilized Candida antarctica lipase (Novozym 435) was studied using trioleoylglycerol (TO) as a model substrate. Optimization of the reactant weight ratio revealed that the 2-MAG reaction yield increased when a larger amount of ethanol was used. These results suggested that Novozym 435 showed strict regiospecificity in an excess amount of ethanol. The process optimization (reaction temperature and reactant molar ratio) and a study of lipase specificity for various substrates were performed. Under the optimized conditions (ethanol/TO molar ratio = 77:1 and 25degreesC), 2-monooleoylglycerol (2-MO) was obtained in more than 98% content among glycerides of the reaction mixture and approximately 88% reaction yield in 4 h. The above reaction conditions were applied for ethanolysis of tridocosahexaenoylglycerol, trieicosapentaenoylglycerol, triarachidonoylglycerol, tri-alpha-linolenoylglycerol, and trilinoleoylglycerol. Reaction yields ranging from 71.9 to 93.7% were obtained in short reaction times (2.5 to 8 h). Purified (>98%) 2-MO and 2-monodocosahexaenoylglycerol (2-MD) were reesterified with caprylic acid by immobilized Rhizomucor miehei lipase (Lipozyme IM) to afford symmetrical structured TAG. At a stoichionnetric ratio of 2-MAG/caprylic acid, 25degreesC and 2-5 mm Hg vacuum, the glyceride composition of the esterification mixture was approximately 95% 1,3-dicapryloyl-2-oleoylglycerol (COC) at 4 h, and 96% 1,3-d dicapryloyl-2-docosahexaenoylglycerol (CDC) at 8 h. The regioisomeric purity of both COC and CDC was 100%.

    DOI: 10.1007/s11746-002-0573-8

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  33. Biosynthetic pathway of diepoxy bicyclic FA from linoleic acid by Clavibacter sp ALA2

    Y Iwasaki, W Brown, CT Hou

    JOURNAL OF THE AMERICAN OIL CHEMISTS SOCIETY   79 巻 ( 4 ) 頁: 369 - 372   2002年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER OIL CHEMISTS SOC A O C S PRESS  

    The biosynthetic pathway of two bicyclic FA, 12:1 7,13:17-diepoxy-9(Z)-octadecenoic acid (DEOA) and 7-hydroxy-12:17,13:17-diepoxy-9(Z)-octadecenoic acid (hDEOA), by Clavibacter sp. ALA2 was investigated, When cultivated with linoleic acid as a substrate, the strain produced 12,13,17-trihydroxy-9(Z)-octadecenoic acid (THOA), DEOA, and hDEOA as well as other FA. To clarify the synthetic route to these bicyclic FA, the strain was cultivated with purified THOA as a starting substrate. THOA was consumed almost completely by the strain with sequential generation of DEOA and hDEOA. Moreover, the strain produced hDEOA when cultivated with purified DEOA. Therefore, it was confirmed that THOA was a precursor of these bicyclic FA and that hDEOA was generated from DEOA. Based on our previously reported result that linoleic acid is first converted to 12,13-dihydroxy-9(Z)-octadecenoic acid (DHOA) and the present results, the overall biosynthetic pathway for the diepoxy bicyclic FA from linoleic acid was postulated as: linoleic acid --> DHOA --> THOA --> DEOA --> hDEOA.

    DOI: 10.1007/s11746-002-0490-x

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  34. Reduction of protein degradation by use of protease-deficient mutants in cell-free protein synthesis system of Escherichia coli

    XP Jiang, K Oohira, Y Iwasaki, H Nakano, S Ichihara, T Yamane

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   93 巻 ( 2 ) 頁: 151 - 156   2002年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SOC BIOSCIENCE BIOENGINEERING JAPAN  

    In an Escherichia coli in vitro transcription/translation system, the degradation of produced proteins is often caused by endogenous proteases from E. coli extracts. To reduce the extent of this degradation, several extracts were prepared from E. coli mutants that genetically lacked DegP, OmpT, or Lon proteases. Then, these extracts were used with C-14-leucine in a system for synthesizing single-chain Fv against gp120 (anti-gp120), and phospholipase D (PLD) of Streptomyces antibioticus. The proteins synthesized in vitro were analyzed by sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis (PAGE) and autoradiography. The use of extracts from mutants that were deficient in the structural genes encoding OmpT and Lon markedly repressed the degradation of anti-gp120. Similarly, extracts from degP- and ompT-deleted mutants were able to significantly stabilize in vitro-synthesized PLD, which otherwise disappeared within 30 min. Such protease-deficient mutants were suggested to be useful for preventing the degradation of heterologous proteins in in vitro systems.

    DOI: 10.1263/jbb.93.151

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  35. Phospholipases in Enzyme Engineering of Phospholipids for Food, Cosmetics and Medical Applications 招待有り

    Yugo Iwasaki and Tsuneo Yamane

    Lipid Biotechnology, (T.M. Kuo and H.W. Gardner ed.), Marcel Deckker Inc. New York (2002).   Chap.20 巻   頁: 417-431   2002年

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    担当区分:筆頭著者   記述言語:英語  

  36. Two-step enzymatic synthesis of docosahexaenoic acid-rich symmetrically structured triacylglycerols via 2-monoacylglycerols

    R Irimescu, K Furihata, K Hata, Y Iwasaki, T Yamane

    JOURNAL OF THE AMERICAN OIL CHEMISTS SOCIETY   78 巻 ( 7 ) 頁: 743 - 748   2001年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER OIL CHEMISTS SOC A O C S PRESS  

    Symmetrically structured triacylglycerols (TG) rich in docosahexaenoic acid (DHA) with caprylic acid (CA) at the outer positions were synthesized enzymatically from bonito oil in a two-step process: (i) ethanolysis of bonito oil TG to 2-monoacylglycerols (2-MG) and fatty acid ethyl esters, and (ii) reesterification of 2-MG with ethyl caprylate. Ethanolysis catalyzed by immobilized Candida antarctica lipase (Novozym 435) yielded 92.5% 2-MG with 43.5% DHA content in 2 h. The 2-MG formed were reesterified with ethyl caprylate by immobilized Rhizomucor miehei lipase (Lipozyme IM) to give structured TG with 44.9% DHA content [based on fatty acid composition with caprylic acid (CA) excluded] in 1 h. The final structured lipids comprised 85.3% TG with two CA residues and one original fatty acid residue, 13% TG with one CA residue and two original fatty acid residues, and 1.7% tricapryloylglycerol (weight percent). The amount of TG with two CA residues and one C-22 residue (22:6 = DHA, 22:5, and 22:4) was 51 wt%. The 1,3-dicapryloyl-2-docosahexaenoylglycerol to 1,2(2,3)-dicapryloyl-3(1)-docosahexaenoylglycerol ratio (based on high-performance liquid chromatography peak area percentages) was greater than 50:1. The recovery of TG as structured lipids after silica gel column purification was approximately 71 %. Ethyl esters and 2-MG formed at 2 h of ethanolysis could be used to determine the positional distribution of fatty acids in the initial TG owing to the high 1,3 -regiospecificity of Novozym 435 and the reduced acyl migration in the system.

    DOI: 10.1007/s11746-001-0336-6

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  37. Utilization of reaction medium-dependent regiospecificity of Candida antarctica lipase (Novozym 435) for the synthesis of 1,3-dicapryloyl-2-docosahexaenoyl (or eicosapentaenoyl) glycerol

    R Irimescu, K Furihata, K Hata, Y Iwasaki, T Yamane

    JOURNAL OF THE AMERICAN OIL CHEMISTS SOCIETY   78 巻 ( 3 ) 頁: 285 - 289   2001年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER OIL CHEMISTS SOC A O C S PRESS  

    A highly efficient enzymatic method for the synthesis of regioisomerically pure 1,3-dicapryloyl-2-docosahexaenoyl glycerol (CDC) in two steps was established. 2-Monoglyceride (2-MG) formation by ethanolysis of tridocosahexaenoylglycerol (DDD) with immobilized Candida antarctica lipase (Novozym 435) as catalyst was the key step of the synthesis. CDC was finally obtained by reesterification of 2-MG with ethylcaprylate (EtC) catalyzed by Rhizomucor miehei lipase (Lipozyme IM). The regiospecificity of Novozym 435 depended on the type of reaction and the initial composition of the reaction medium. It displayed strict 1,3-regiospecifcity for ethanolysis at a high excess of ethanol in the reaction mixture although it displayed no regiospecificity in transesterification and esterification reactions. The highest yield of CDC (85.4%) was obtained by ethanolysis at a 4:1 weight ratio of ethanol/ DDD for 6 h followed by reesterification at a 20:1 molar ratio of EtC/initial DDD for 1.5 h. The regioisomeric purity of CDC was 100%. Good results were obtained also for the synthesis of 1,3-dicapryloyl-eicosapentaenoylglycerol (CEC) by the same method: 84.2% yield and 99.8% regioisomeric purity at the same reactant ratios as above. The yield of the reesterification step and the regioisomeric purity of the product were influenced by the molar ratio of the reactants for both CDC and CEC syntheses: higher excess of EtC favored higher yields and regioisomeric purity of the products.

    DOI: 10.1007/s11746-001-0258-3

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  38. Comparison of acyl donors for lipase-catalyzed production of 1,3-dicapryloyl-2-eicosapentaenoylglycerol 査読有り

    R Irimescu, K Hata, Y Iwasaki, T Yamane

    JOURNAL OF THE AMERICAN OIL CHEMISTS SOCIETY   78 巻 ( 1 ) 頁: 65 - 70   2001年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER OIL CHEMISTS SOC A O C S PRESS  

    Synthesis of 1,3-dicapryloyl-2-eicosapentaenoyl glycerol (CEC) catalyzed by Lipozyme IM (immobilized Rhizomucor miehui lipase) was performed by interesterification of trieicosapentaenoylglycerol (ECD with caprylic acid ICA) (acidolysis) and EEE with ethyl caprylate (EtC) (interesterification). Both methods involved two steps: (i) transesterification at an optimized water content and temperature for the high yield conversion of the substrate to CEC, 1-capryloyl-2-eicosaptatacnoylglycerol (CEOH) and 2-eicosapentaenoylglycerol (OHEOH), and (ii) reesterification of CEOH and OHEOH to CEC by water removal under reduced pressure. Interesterification had clear advantages over acidolysis. The reaction rates for interesterification were higher and the reaction times shorter. The final yield of CEC by interesterification was higher, and the extent of acyl migration, indicated by the tricapryloylglycerol content, was lower. The disadvantage of the higher price of EtC used for interesterification (approximately 10 times higher than the price of CA) was overcome by synthesizing it directly in the same reaction vessel prior to the interesterification step. EtC was rapidly synthesized by esterification of CA with ethanol in high yield (92% obtained in 2.5 h). The amount of water added to the reaction mixture and the reaction temperature influenced the yields of CEC, CEOH, and OHEOH in the transesterification step for both interesterification and acidolysis methods. The regioisomeric purity of CEC was 100% for both methods at temperatures of 40 degreesC or less. The highest yield of CEC (81%) was obtained for the interesterification of EEE with EtC, formed directly in the same reaction vessel, at a CA/EEE molar ratio of 20:1 and 30 degreesC.

    DOI: 10.1007/s11746-001-0221-3

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  39. Optical resolution of asymmetric triacylglycerols by chiral-phase high-performance liquid chromatography

    Y Iwasaki, M Yasui, T Ishikawa, R Irimescu, K Hata, T Yamane

    JOURNAL OF CHROMATOGRAPHY A   905 巻 ( 1-2 ) 頁: 111 - 118   2001年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER SCIENCE BV  

    A simple method for direct optical resolution of some asymmetric triacylglycerols (TGs) has been established. The method employs chiral-phase high-performance liquid chromatography (HPLC). An enantiomeric pair of TGs comprising 1-eicosapentaenoyl-2,3- dicapryroyl-sn-glycerol (ECC) and 1,2-dicapryroyl-3-eicosapentaenoyl-sn-glycerol (CCE) was resolved on a CHIRALCEL OF(TM) or on a CHIRALCEL OD(TM) column. The separation of another pair of asymmetric TGs, 1-docosahexaenoyl-2,3-dicapryroyl-sn-glycerol (DCC) and 1,2-dicapryroyl-3 -docosahexaenoyl-sn-glycerol (CCD), was achieved with the CHIRALCEL OD column. The chiral-phase HPLC method in combination with silver-ion HPLC and high-temperature gas chromatography was used for monitoring two interesterification reactions, whose products were chiral TGs. Interesterification of tricapryloylglycerol with ethyleicosapentaenoate or with ethyldocosahexaenoate was performed using Rhizomucor miehei lipase as the catalyst. The products targeted were the asymmetric pair of TGs, ECC and CCE or DCC and CCD. The amounts of sn-l-substituted products (ECC or DCC) were greater than their sn-3-substituted counterparts (CCE or CCD) throughout the reaction period, suggesting that R. miehei lipase had a stereopreference towards the sn-1 position over the sn-3 position. (C) 2001 Elsevier Science B.V. All rights reserved.

    DOI: 10.1016/S0021-9673(00)00989-4

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  40. 構造脂質の酵素合成 招待有り

    "岩崎雄吾,山根恒夫"

    オレオサイエンス   1 巻   頁: 825-833   2001年

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    記述言語:日本語  

  41. Enzymatic synthesis of structured lipids 査読有り

    Y Iwasaki, T Yamane

    JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC   10 巻 ( 1-3 ) 頁: 129 - 140   2000年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER SCIENCE BV  

    Lipases are powerful tools for the syntheses of structured lipids (SLs) which are triacylglycerols (TGs) having particular fatty acid (FA) residues at specific positions. With respect to the number of FA species and their distribution in glycerol molecule, TGs are classified into several types; AAA, ABA, AAB, and ABC types. Among them, AAA-type TGs can be synthesized from FA and glycerol either chemically or enzymatically. Even at stoichiometric mixture of the substrates, almost complete reaction is possible. Syntheses of the other types of TGs require positionally specific reactions, for which the use of regiospecific lipases are effective. ABA-type SLs are synthesized by (1) sn-1,3-position-specific lipase-catalyzed interesterification of two different TGs, (2) sn-1,3-position-specific lipase-catalyzed acyl exchange of TG with FA or with FA ethylester (FAEt), (3) sn-1,3-position-specific lipase-catalyzed acylation of glycerol with FA giving symmetric 1,3-diacyl-sn-glycerol, followed by chemical acylation at sn-2 position. ABB-type SL is prepared by lipase-catalyzed monosubstitution at either sn-1 or -3 position of TG with FA or with FAEt, avoiding formation of disubstituted by-product. Stereopreference to sn-1 position over sn-3 position of a certain kind of lipase enables the syntheses of chiral ABB- and ABC-type TGs. (C) 2000 Elsevier Science B.V. All rights reserved.

    DOI: 10.1016/S1381-1177(00)00120-X

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  42. Enzymatic synthesis of 1,3-dicapryloyl-2-eicosapentaenoylglycerol

    R Irimescu, M Yasui, Y Iwasaki, N Shimidzu, T Yamane

    JOURNAL OF THE AMERICAN OIL CHEMISTS SOCIETY   77 巻 ( 5 ) 頁: 501 - 506   2000年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER OIL CHEMISTS SOC A O C S PRESS  

    1,3-Dicapryloyl-2-eicosapentaenoylglycerol (CEC) was synthesized by interesterification of trieicosapentaenoylglycerol (EEE) with ethyl caprylate (EtC) catalyzed by Lipozyme(TM). After some of the reaction conditions were optimized, the maximal molar content of CEC in the glycerides of the reaction mixture was 91%. Among the parameters studied in the optimization, the critical ones were: (i) the water content, which influenced the conversion of EEE to CEC and 1-capryloyl-2-eicosapentaenoylglycerol (CEOH), and (ii) the timing of water removal under reduced pressure for the reesterification of CEOH to form CEC. The complete synthesis of CEC from ethyl eicosapentaenoate (EtE) was performed in three steps: (i) hydrolysis of BE to free eicosapentaenoic acid (EPA), (ii) esterification of glycerol with EPA to form EEE, and (iii) interesterification of EEE with Etc under the optimized conditions. The first two steps were catalyzed by Novozym(TM) and the third by Lipozyme(TM). The total yield over all the steps was 88%, and no purification of the intermediates was necessary. The regioisomeric purity of the product was 100% by silver-ion high-pressure liquid chromatography.

    DOI: 10.1007/s11746-000-0080-y

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  43. Metabolism of 1-acyl-2-oleoyl-sn-glycero-3-phosphoethanolamine in castor oil biosynthesis

    JT Lin, KM Lew, JM Chen, Y Iwasaki, TA McKeon

    LIPIDS   35 巻 ( 5 ) 頁: 481 - 486   2000年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SPRINGER HEIDELBERG  

    We have examined the role of 2-oleoyl-PE (phosphatidylethanolamine) in the biosynthesis of triacylglycerols (TAG) by castor microsomes. In castor microsomal incubation, the label from C-14-oleate of 1-palmitoyl-2-[1-C-14]oleoyl-sn-glycero-3-phosphoethanolamine is incorporated into TAG containing ricinoleate. The enzyme characteristics, such as optimal pH, and the effect of incubation components of the oleoyl-12-hydroxylase using 2-oleoyl-PE as incubation substrate are similar to those for 2-oleoyl-PC (phosphatidylcholine). However, compared to 2-oleoyl-PC, 2-oleoyl-PE is a less efficient incubation substrate of oleoyl-12-hydroxylase in castor microsomes. Unlike 2-oleoyl-PC, 2-oleoyl-PE is not hydroxylated to 2-ricin-oleoyl-PE by oleoyl-12-hydroxylase and is not desaturated to 2-linoleoyl-PE by oleoyl-12-desaturase. We have demonstrated the conversion of 2-oleoyl-PE to 2-oleoyl-PC and vice versa. The incorporation of label from 2-[C-14]oleoyl-PE into TAC occurs after its conversion to 2-oleoyl-PC, which can then be hydroxylated or desaturated. We detected neither PE-N-monomethyl nor PE-N,N-dimethyl, the intermediates from PE to PC by N-methylation. The conversion of 2-oleoyl-PE to 2-oleoyl-PC likely occurs via hydrolysis to 1,2-diacyl-sn- glycerol by phospholipase C and then by cholinephosphotransferase. This conversion does not appear to play a key role in driving ricinoleate into TAG.

    DOI: 10.1007/s11745-000-547-5

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  44. Importance of disulfide bridge formation on folding of phospholipase D from Streptomyces antibioticus

    Y Iwasaki, T Nishiyama, Y Kawarasaki, H Nakano, T Yamane

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   89 巻 ( 5 ) 頁: 506 - 508   2000年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SOC BIOSCIENCE BIOENGINEERING JAPAN  

    The effects of redox conditions on the folding of phospholipase D (PLD) of Streptomyces antibioticus were investigated. Although the enzyme was very stable even in the presence of 1.0 M guanidinehydrochrolide (Gdn-HCl), the coexistence of dithiothreitol (DTT) and Gdn-HCl inactivated the enzyme completely. The inactivated enzyme recovered its activity by dialysis in which DTT was removed prior to Gdn-HCl, whereas its activity was not recovered when Gdn-HCl was removed prior to DTT. In vitro protein synthesis was used for further analyses of the folding process. Active PLD was synthesized In the absence of DTT. The activity increased as the protein synthesis proceeded. In contrast, inactive PLD was synthesized In the presence of DTT. The inactive PLD could not be effectively activated by simple removal of the reductant, while incubation with Gdn-HCl and subsequent removal of DTT followed by that of Gdn-HCl was a much more effective method for the synthesis of active enzymes. From these results, it is suggested that: (i) PLD contains disulfide bridge(s), which is (are) necessary for maintaining its tertiary structure, (ii) correct formation of the disulfide bridge(s) is a critical step in the early stage of the (re)folding process, and (iii) the disulfide bridge(s) further facilitate the folding process, resulting in the synthesis of the active enzymes with the correct structure.

    DOI: 10.1016/S1389-1723(00)89107-0

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  45. In vitro analysis of roles of a disulfide bridge and a calcium binding site in activation of Pseudomonas sp strain KWI-56 lipase

    JH Yang, K Kobayashi, Y Iwasaki, H Nakano, T Yamane

    JOURNAL OF BACTERIOLOGY   182 巻 ( 2 ) 頁: 295 - 302   2000年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER SOC MICROBIOLOGY  

    The expression of lipase from Pseudomonas sp. strain KWI-56 (recently reclassified as Burkholderia cepacia) had been found to be dependent on an activator gene tact) downstream of its structural gene (lip). In this work, the mature lipase was synthesized in an enzymatically active form with a cell-free Escherichia coli S30 coupled transcription-translation system by expressing a recombinant lipase gene (rlip! encoding the mature lipase in the presence of its purified activator or by coexpression of rlip and act. The in vitro expression systems were used for studying the folding process of the lipase. The addition of dithiothreitol in the expression systems decreased the activity dramatically without affecting the synthesis level of the lipase, whereas the in vitro-synthesized active lipase was relatively stable even in the presence of dithiothreitol. This phenomenon was further investigated by constructing mutant lipase genes only in vitro by PCR without gene cloning. Replacements of cysteine residues (Cys190 and Cys270) forming a sole putative disulfide bond to serine residues decreased the lipase activity greatly, suggesting that the disulfide bond was essential for the proper folding of the lipase. In addition, replacing Asp242 and Asp288, which were deduced to be part of a Ca2+ binding site, also greatly decreased the activities of the in vitro-synthesized lipases. The role of the Ca2+ binding site in the activation of the lipase is also discussed.

    DOI: 10.1128/JB.182.2.295-302.2000

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  46. In vitro analysis of roles of a disulfide bridge and a calcium binding site in activation of Pseudomonas sp. strain KWI-56 lipase. 査読有り

    Jun-Hao Yang, Koei Kobayashi, Yugo Iwasaki, Hideo Nakano and Tsuneo Yamane

    J. Bacteriol.   182 巻   頁: 295-302   2000年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

  47. Metabolism of 1-acyl-2-oleoyl-sn-glycero-3-phosphoethanolamine in castor oil biosynthesis. 査読有り

    Jiann-Tsyh Lin, Karen M. Lew, Jennifer M. Chen, Yugo Iwasaki and Thomas A. McKeon

    Lipids   35 巻   頁: 481-486   2000年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

  48. Importance of disulfide bridge formation on folding of phospholipase D from Streptomyces antibioticus 査読有り

    Yugo Iwasaki, Tatsuaki Nishiyama, Yasuaki Kawarasaki, Hideo Nakano and Tsuneo Yamane

    J. Biosci. Bioeng.   89 巻   頁: 506-508   2000年

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    担当区分:筆頭著者   記述言語:英語   掲載種別:研究論文(学術雑誌)  

  49. Enzymatic synthesis of 1,3-dicapryloyl-2-eicosapentaenoylglycerol 査読有り

    Roxana Irimescu, Mamoru Yasui, Yugo Iwasaki, Nobuyoshi Shimidzu and Tsuneo Yamane

    J. Am. Oil Chem. Soc.   77 巻   頁: 501-506   2000年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

  50. 機能性脂質とバイオテクノロジー

    山根恒夫, 岩崎雄吾

    バイオサイエンスとバイオインダストリー   58 巻   頁: 11 - 16   2000年

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    記述言語:日本語   掲載種別:研究論文(学術雑誌)  

  51. Location of the catalytic nucleophile of phospholipase D of Streptomyces antibioticus in the C-terminal half domain

    Y Iwasaki, S Horiike, K Matsushima, T Yamane

    EUROPEAN JOURNAL OF BIOCHEMISTRY   264 巻 ( 2 ) 頁: 577 - 581   1999年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:BLACKWELL SCIENCE LTD  

    Phospholipase D (PLD) of Streptomyces antibioticus was labelled with fluorescent-labelled substrate, 1-hexanoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)-amino]hexanoyl}-sn-glycero-3-phosphocholine, when it was incubated with the substrate and the reaction followed by SDS/PAGE. Mutant enzymes lacking the catalytic activity were not labelled under the same conditions, indicating that labelling of the PLD occurred as the result of its catalytic action. This confirmed that the labelled protein was the phosphatidyl PLD intermediate. PLDs contain two copies of the highly conserved catalytic HxKxxxxD (HKD) motif. Therefore, two protein fragments were separately prepared with recombinant strains of Escherichia coli. One of the fragments was the N-terminal half of the intact PLD containing one HKD motif, and the other was the C-terminal half with the other motif. An active enzyme was reconstructed from these two fragments, and therefore designated fragmentary PLD (fPLD). When fPLD was subjected to the labelling experiment, only the C-terminal half was labelled. Therefore, it was concluded that the catalytic nucleophile that bound directly to the phosphatidyl group of the substrate was located on the C-terminal half of PLD, and that the N-terminal half did not contain such a nucleophile.

    DOI: 10.1046/j.1432-1327.1999.00669.x

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  52. Enzymatic synthesis of symmetrical 1,3-diacylglycerols by direct esterification of glycerol in solvent-free system

    R Rosu, M Yasui, Y Iwasaki, T Yamane

    JOURNAL OF THE AMERICAN OIL CHEMISTS SOCIETY   76 巻 ( 7 ) 頁: 839 - 843   1999年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER OIL CHEMISTS SOC A O C S PRESS  

    1,3-Diacylglycerols were synthesized by direct esterification of glycerol with free fatty acids in a solvent-free system. Free fatty acids with relatively low melting points (<45 degrees C) such as unsaturated and medium-chain saturated fatty acids were used. With stoichiometric ratios of the reactants and water removal by evaporation at 3 mm Hg vacuum applied at 1 h and thereafter, the maximal 1,3-diacylglycerol content in the reaction mixture was: 84.6% for 1,3-dicaprylin, 84.4% For 1,3-dicaprin, 74.3% for 1,3-dilinolein, 71.7% for 1,3-dieicosapentaenoin, 67.4% for 1,3-dilaurin, and 61.1% for 1,3-diolein. Some of the system's parameters (temperature, water removal, and molar ratio of the reactants) were optimized for the production of 1,3-dicaprylin, and the maximal yield reached 98%. The product was used for the chemical synthesis of 1,3-dicapryloyl-2-eicosapentaenoylglycerol. The yield after purification was 42%, and the purity of the triacylglycerol was 98% (both 1,3-dicapryloyl-2-eicosapentaenoylglycerol and 1,2-dicapryloyl-3-eicosapentaenoylglycerol included) by gas chromatographic analysis, of which 90% was the desired structured triacylglycerol (1,3-dicapryloyl-2-eicosapentaenoylglycerol) as determined by silver ion high-performance liquid chromatographic analysis.

    DOI: 10.1007/s11746-999-0074-7

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  53. Use of isopropanol as a modifier in a hexane-acetonitrile based mobile phase for the silver ion HPLC separation of positional isomers of triacylglycerols containing long chain polyunsaturated fatty acids

    JJ Han, Y Iwasaki, T Yamane

    HRC-JOURNAL OF HIGH RESOLUTION CHROMATOGRAPHY   22 巻 ( 6 ) 頁: 357 - 361   1999年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY-V C H VERLAG GMBH  

    A high resolution approach to silver ion HPLC was studied for the separation of positional isomers of triacylglycerols (TAGs) containing long chain polyunsaturated fatty acids (PUFA) such as eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosapentaenoic acid (DPA) in enzymatically synthesized structured TAGs. Isopropanol was used as a novel modifier in a hexane-acetonitrile based mobile phase for silver ion HPLC. Peak identification was based on HPLC-mass Spectroscopy and selectivities of lipases. Positional isomers of TAGs containing one molecule of EPA, DHA, or DPA with saturated fatty acids (FAs) such as caprylic acid and palmitic acid were separated within 13 min using a gradient of hexane-isopropanol-acetonitrile as mobile phase. TAGs containing two or more EPA, DHA, or DPA were also separated from each other within 25 min, but their positional isomers were unresolved. The retention characteristics of the TAG were found to be related to the number of carbon atoms in the FAs present in addition to the number of double bonds and their isomeric configuration. One isomer with an unsaturated FA in the sn-2 position eluted faster than the other with the unsaturated FA in the sn-1 or 3 position. Species with longer chain FAs attached to TAGs with the same degree of unsaturation eluted faster than those that have shorter chain FAs, For example, docosapentaenoylhexadecanoyloctanoin (DPA/C16/C8) was eluted faster than dioctanoyldocosapentaenoin (DPA/C8/C8).

    DOI: 10.1002/(SICI)1521-4168(19990601)22:6<357::AID-JHRC357>3.0.CO;2-#

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  54. Enzymatic synthesis of structured lipids from single cell oil of high docosahexaenoic acid content 査読有り

    Y Iwasaki, JJ Han, M Narita, R Rosu, T Yamane

    JOURNAL OF THE AMERICAN OIL CHEMISTS SOCIETY   76 巻 ( 5 ) 頁: 563 - 569   1999年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER OIL CHEMISTS SOC A O C S PRESS  

    The lipase-catalyzed acidolysis of a single-cell oil (SCO) containing docosahexaenoic acid (DHA) and docosapentaenoic acid (DPA) with caprylic acid (CA) was investigated. The targeted products were structured lipids containing CA residues at the sn-1 and -3 positions and a DHA or DPA residue at the sn-2 position of glycerol. Rhizomucor miehei lipase (RML) and Pseudomanas sp. KWI-56 lipase (PSL) were used as the biocatalysts, When PSL was used &gt; 60 mol% of total SCO fatty acids (FA) were exchanged with CA, with DHA and DPA as well as the other saturated FA being exchanged. The content of the triacylglycerols (TG) containing two CA and one DHA or DPA (number of carbon atoms = 41, i.e., C-41) residue was high (36%), and the isomer with the desired configuration (unsaturated FA residue at the sn-2 position) represented 77-78% of C-41. In the case of RML, CA content reached only 23 mol% in the TG. A large amount of DHA and DPA residues remained unexchanged with RML, so that the resulting oil was rich in TC species containing two or three DHA or DPA residues (46%). TG C-41 amounted to 22%, almost all of which had the desired configuration. This result suggested that the difference in the degree of acidolysis by the two enzymes was due to their different selectivity toward DHA and DPA, as well as the difference in their positional specificities.

    DOI: 10.1007/s11746-999-0005-7

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  55. Crystallization and preliminary x-ray diffraction studies of phospholipase D from Streptomyces antibioticus 査読有り

    Atsuo Suzuki, Kouji Kakuno, Yugo Iwasaki, Tsuneo Yamane, Takashi Yamane

    Acta Crystallographica Section D: Biological Crystallography   55 巻 ( 1 ) 頁: 317 - 319   1999年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Blackwell Publishing Ltd  

    Phospholipase D (E.C. 3.1.4.4) from Streptomyces antibioticus has been crystallized in six crystal forms using the hanging-drop vapour-diffusion method. The type III and V crystals belong to monoclinic and hexagonal systems, respectively. All of the other crystal forms, types I, II, IV and VI, belong to orthorhombic space group P212121. Of these four types, the type VI crystals are suitable for X-ray structure determination. Crystal data for type VI crystals are: a = 50.1, b = 98.7, c = 107.6 Å, V = 532 100 Å3, Z = 4 and V(m) = 2.47 Å Da-1. Type VI crystals diffract to at least 2.3 Å resolution. A total of 11 295 independent reflections to 3 Å resolution have been collected from a type VI crystal using a conventional X-ray source, and its structural analysis is currently being conducted using isomorphous replacement methods.

    DOI: 10.1107/S0907444998010592

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  56. Monitoring of lipase-catalyzed transesterification between eicosapentaenoic acid ethylester and tricaprylin by silver ion high performance liqiud chromatography and high temperature gas chromatography. 査読有り

    Jeong-Jun Han, Yugo Iwasaki and Tsuneo Yamane

    J. Am. Oil Chem. Soc.   76 巻   頁: 31-39   1999年1月

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    担当区分:筆頭著者   記述言語:英語   掲載種別:研究論文(学術雑誌)  

  57. Enzymatic synthesis of symmetrical 1,3-diacylglycerols by direct esterification of glycerol in solvent-free system. 査読有り

    Roxana Rosu, Mamoru Yasui, Yugo Iwasaki, and Tsuneo Yamane

    J. Am. Oil Chem. Soc.   76 巻   頁: 839-843   1999年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

  58. Intensification of lipase performance in a transesterification reaction by immobilization on CaCO3 powder

    R Rosu, Y Iwasaki, N Shimizu, N Doisaki, T Yamane

    JOURNAL OF BIOTECHNOLOGY   66 巻 ( 1 ) 頁: 51 - 59   1998年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER SCIENCE BV  

    Pseudomonas lipase was used as catalyst for the transesterification of docosahexaenoic acid ethyl ester with glycerol under low pressure in a solvent free system. The biocatalyst performance was intensified greatly by prior adsorption of lipase on very fine CaCO3 powder. CaCO3 particles played the role of a surfactant and of an enzyme transporter to the glycerol-ethyl ester interface. Microscopic analysis of the emulsified reaction mixtures revealed that CaCO3 particles stabilized the droplets of substrate by adsorption on their surface. The reaction rate, when the immobilized enzyme was used, was five times higher than that of the reaction performed with dissolved free lipase, due to the larger interfacial area available for the enzyme action. The reaction with immobilized enzyme reached equilibrium at 96% consumption of ethyl ester after 8 h of reaction time. In contrast, the reaction with free enzyme was far from equilibrium at only 43% conversion at the same reaction time. The prior immobilization of lipase on CaCO3 proved to be necessary for the operational stability of the catalyst by protecting the enzyme against inactivation. (C) 1998 Elsevier Science B.V. Al rights reserved.

    DOI: 10.1016/S0168-1656(98)00156-4

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  59. Enzymatic synthesis of glycerides from DHA-enriched PUFA ethyl ester by glycerolysis under vacuum

    R Rosu, Y Iwasaki, N Shimidzu, N Doisaki, T Yamane

    JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC   4 巻 ( 4 ) 頁: 191 - 198   1998年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER SCIENCE BV  

    Pseudomonas lipase immobilized on CaCO3 powder was used for the glycerolysis of n - 3 polyunsaturated fatty acid ethyl esters to prepare nutritionally valuable glycerides. The initial ethyl ester contained 65 or 99% docosahexaenoic acid ethyl ester (DHAEE) which is very unstable and readily oxidized. The process performance was intensified by: (1) using Pseudomonas lipase which has good specificity for docosahexaenoic acid (DHA), (2) shifting the reaction equilibrium by evaporation of the resulted ethanol under vacuum and (3) enhancing the enzyme operational stability by immobilization on CaCO3 powder. Under these conditions, over 90% conversion of DHAEE was achieved in 5 h and the oxidative deterioration of DHA was avoided. The final product contained 53% partial glyceride and, thus, had good emulsifying power. The catalyst was reused 5 times showing a very good stability in this system. Other lipases were tried for this reaction and different glyceride compositions were obtained depending on the enzyme specificity for the 1(3)-position of glycerol. (C) 1998 Elsevier Science B.V. All rights reserved.

    DOI: 10.1016/S1381-1177(97)00035-0

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  60. Two distinct phosphatidylinositol-specific phospholipase Cs from Streptomyces antibioticus

    Y Iwasaki, Y Tsubouchi, A Ichihashi, H Nakano, T Kobayashi, H Ikezawa, T Yamane

    BIOCHIMICA ET BIOPHYSICA ACTA-LIPIDS AND LIPID METABOLISM   1391 巻 ( 1 ) 頁: 52 - 66   1998年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER SCIENCE BV  

    Two phosphatidylinositol-specific phospholipase C (PI-PLC) genes from Streptomyces antibioticus were cloned by a shotgun method using Streptomyces lividans TK24 as a host. The genes of the two PI-PLCs (named as PLC1 and PLC2) were adjoined and opposite in the direction of transcription/translation. Both of them were confirmed to be expressed in S. antibioticus. The two enzymes were different in the following properties, (i) PLC2 had considerable sequence similarity to other bacterial PI-PLCs, while PLC1 had a short stretch that was similar to PI-PLCs of eukaryotes rather than the other bacterial enzymes. (ii) PLC1 was Ca2+-dependent, whereas PLC2 was not. (iii) PLC1 generated myo-inositol-1-phosphate and myo-inositol-1:2-cyclic phosphate simultaneously from PI, but PLC2 showed sequential formation of them. (iv) PLC2 has GPI-anchor-degrading activity while PLC1 does not have. Both enzymes did not hydrolyze phosphatidylcholine, phosphatidylinositol-4-monophosphate and phosphatidylinositol-4,5-bisphosphate. Both PLC1 and PLC2 contained two histidine residues that might be catalytic residues. PLC1 has residues that possibly form a Ca2+-binding site. Then it was suggested that both PLC1 and PLC2 act according to the catalytic mechanism using the two histidine residues as proposed in both eukaryotic and prokaryotic enzymes, but that PLC1 has a more 'eukaryotic' mechanism in which Ca2+ participates than that of the Ca2+-independent bacterial enzymes. Thus, we propose that PLC2 is a conventional 'bacteria-type' enzyme, while PLC1 is more closely related to the eukaryotic enzymes rather than the bacterial enzymes. (C) 1998 Elsevier Science B.V.

    DOI: 10.1016/S0005-2760(97)00191-4

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  61. Enzymatic synthesis of glycerides from DHA-enriched PUFA ethyl ester by glycerolysis uer vacuum. 査読有り

    Rosu, R., Iwasaki, Y., Shimidzu, N., Doisaki N. and Yamane, T.

    J. Mol. Cat.B: Enz   4 巻   頁: 191-198   1998年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

  62. Multiple intensified performance of an enzyme-catalyzed reaction in organic medium

    T Yamane, Y Iwasaki, R Roxana, N Shimidzu, N Doisaki

    ENZYME ENGINEERING XIV   864 巻   頁: 171 - 179   1998年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:NEW YORK ACAD SCIENCES  

    A lipase-catalyzed glycerolysis reaction (a transesterification between polyunsaturated fatty acid ethyl ester [PUFA] and glycerol) was investigated. Its performance was multiply intensified by (1) using a lipase having high specific activity, high activity in organic solvent, and high tolerance in organic solvent; (2) immobilization on fine CaCO3 powder (cheap and safe material, easy physical adsorption method of immobilization, reusable); (3) reaction in vacuo resulting in 100% conversion and effective avoidance of oxidative deterioration of PUPA; (4) high volumetric productivity because of no use of solvent; and (5) no need of further separation and purification of the oil product. It is emphasized that performance of biocatalytic reactions in organic media should be enhanced manifold for industrial implementation.

    DOI: 10.1111/j.1749-6632.1998.tb10299.x

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  63. Insertion of stabilizing loci in vectors of T7 RNA polymerase-mediated Escherichia coli expression systems: A case study on the plasmids involving foreign phospholipase D gene

    N Mishima, K Mizumoto, Y Iwasaki, H Nakano, T Yamane

    BIOTECHNOLOGY PROGRESS   13 巻 ( 6 ) 頁: 864 - 868   1997年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER CHEMICAL SOC  

    Plasmids carrying stabilizing loci were used in the expression of phospholipase D (PLD) gene fused with pelB signal sequence by a recombinant strain of Escherichia coli BL21 (DE3) using T7 RNA polymerase mediated expression system. By checking the living cell number and the percentage of the plasmid-bearing cells, it was found that the plasmids involving PLD gene were not stable under noninduced conditions and that, after the induction, the number of plasmid-bearing cells were rapidly decreased to almost zero. Then, a biologically stabilizing locus such as par B, ccd, or par was inserted into the plasmids. The newly constructed plasmids were maintained very stably in the recombinant cells until the cells were induced. However, after the induction, almost all the recombinant cells were rapidly killed due to highly toxic PLD. Using the best one of the stabilized PLD-expressing plasmids, PLD production was improved 2-fold.

    DOI: 10.1021/bp970084o

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  64. Repeated use of immobilized lipase for monoacylglycerol production by solid-phase glycerolysis of olive oil

    R Rosu, Y Uozaki, Y Iwasaki, T Yamane

    JOURNAL OF THE AMERICAN OIL CHEMISTS SOCIETY   74 巻 ( 4 ) 頁: 445 - 450   1997年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER OIL CHEMISTS SOC  

    By using immobilized lipase for production of monoacylglycerol (MAG) by solid-phase glycerolysis of fats and oils, the enzyme could be recovered easily from the reaction mixture and recycled to reduce the cost of the catalyst. Several support materials (CaCO3, CaSO4 . 2H(2)O, Ca2P2O7, and Celite) were screened for immobilization of Pseudomonas sp. lipase by adsorption and tested for solid-phase glycerolysis of olive oil. Immobilization made the reuse of enzyme feasible. CaCO proved to be the best support: 90% MAC (wt% in the glycerol-free reaction mixture after 72 h of reaction rime) was obtained until the fifth use, 80% after the seventh use, and 60% after the tenth use. The same support was found suitable for immobilization of two other bacterial lipases from Chromobacterium viscosum and Pseudomonas pseudoalkali.

    DOI: 10.1007/s11746-997-0104-2

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  65. Insertion of stabilizing loci in vectors of T7 RNA polymerase-mediated Escherichia coli expression systems: A case study on the plasmids involving foreign phospholipase D gene. 査読有り

    Naoto Mishima, Kousaku Mizumoto, Yugo Iwasaki, Hideo Nakano and Tsuneo Yamane

    Biotechnol. Prog.   13 巻   頁: 864-868   1997年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

  66. リン脂質類の酵素変換 招待有り

    山根恒夫, 岩崎雄吾

    油化学   44 巻   頁: 875-882   1995年1月

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    担当区分:筆頭著者   記述言語:日本語  

  67. EXTRACELLULAR PRODUCTION OF PHOSPHOLIPASE-D OF STREPTOMYCES-ANTIBIOTICUS USING RECOMBINANT ESCHERICHIA-COLI 査読有り

    Y IWASAKI, N MISHIMA, K MIZUMOTO, H NAKANO, T YAMANE

    JOURNAL OF FERMENTATION AND BIOENGINEERING   79 巻 ( 5 ) 頁: 417 - 421   1995年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SOC BIOSCIENCE BIOENGINEERING JAPAN  

    The phospholipase D (PLD)-encoding gene of Streptomyces antibioticus fused with the pectate lyase B (PelB) signal sequence was expressed in recombinant Escherichia coli under the control of the T7 lac promoter, Most of the PLD activity was detected in the culture supernatant. The N-terminal amino acid sequence of the recombinant PLD was identical to that of the wild-type PLD, suggesting that the processing of the PelB signal peptide occurred correctly and that the PLD produced by E. coli was identical to the wild-type enzyme. The production of PLD was improved by genetic and fermentation techniques. About 3 mg of PLD/l medium was obtained under optimal conditions, comparable to the amount of PLD produced by S. antibioticus. The results of analysis of the distributions of intracellular marker enzymes suggested that neither cell lysis nor periplasmic disruption was the cause of the secretion of PLD.

    DOI: 10.1016/0922-338X(95)91254-3

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  68. PHOSPHOLIPASE-D FROM STREPTOMYCES-ANTIBIOTICUS - CLONING, SEQUENCING, EXPRESSION, AND RELATIONSHIP TO OTHER PHOSPHOLIPASES 査読有り

    Y IWASAKI, F NAKANO, T YAMANE

    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY   42 巻 ( 2-3 ) 頁: 290 - 299   1994年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SPRINGER VERLAG  

    The extracellular phospholipase D (PLD) gene from Streptomyces antibioticus was cloned, sequenced, and expressed in Escherichia coli. Analysis of DNA sequence data revealed a putative ribosome-binding site and an open reading frame encoding a 556-amino-acid protein that included amino acid sequences obtained from the purified enzyme. The protein was expressed in an insoluble form in E. coli, but reacted with antibody against PLD. After solubilization of the protein with guanidine-HCl and 2-mercaptoethanol, subsequent dialysis restored the PLD activity. Comparison of the nucleotide sequence data with the N-terminal protein sequence indicates that this secreted protein is synthesized as a larger precursor with a 47-amino-acid N-terminal extension to the mature enzyme of 509 amino acids. The amino acid sequence of the S. antibioticus PLD was extensively compared with other PLDs and phospholipase C (PLC). The deduced amino acid sequence of the cloned PLD was highly homologous to PLDs from S. acinomyceticus and Streptomyces sp., and contained a conserved region with S. chromofuscus PLD. From comparisons of the structural similarity and properties of the various PLDs, a classification of PLDs into two subgroups has been proposed and the highly conserved region designated tentatively region X(PLD), which may be important in the catalytic function, has been identified. The homology comparison between our PLD and phosphatidylinositol-specific phospholipase C (PI-PLC) is also discussed.

    DOI: 10.1007/s002530050252

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  69. PURIFICATION AND PROPERTIES OF PHOSPHATIDYLINOSITOL-SPECIFIC PHOSPHOLIPASE-C FROM STREPTOMYCES-ANTIBIOTICUS 査読有り

    Y IWASAKI, S NIWA, H NAKANO, T NAGASAWA, T YAMANE

    BIOCHIMICA ET BIOPHYSICA ACTA-LIPIDS AND LIPID METABOLISM   1214 巻 ( 3 ) 頁: 221 - 228   1994年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER SCIENCE BV  

    A novel type of phosphatidylinositol-specific phospholipase C (PI-PLC) was purified from culture supernatant of a strain of Actinomycetales, Streptomyces antibioticus. The purified enzyme showed a single band on native polyacrylamide gel electrophoresis (native PAGE) with a molecular weight of 32 kDa, but showed two polypeptides, named alpha- and beta-peptides, on sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) with molecular weights of 23 kDa and 15 kDa, respectively. From the results of both electrophoretic analysis and N-terminal amino acid sequencing, it was estimated that the enzyme was composed of alpha- and beta-peptides. The enzyme could hydrolyze phosphatidylinositol, but not any other glycerophospholipids. The enzyme had pH and temperature optima at around 7.0 and 30 degrees C, respectively, and was stable up to 50 degrees C when incubated at pH 8.0 for 30 min. The PI-PLC was strongly activated by SDS, sodium deoxycholate (SDC) and diethyl ether, but not by Triton X-100, and inhibited by cetylpyridinium chloride (CPC). The enzyme was activated a little by Ca2+ and was inhibited completely by a chelating agent such as ethylenediaminetetraacetic acid (EDTA) and glycoletherdiaminetetraacetic acid (EGTA). Their inhibitions were restored by the addition of Ca2+, suggesting that a certain amount of Ca2+ is essential for the enzymatic activity.

    DOI: 10.1016/0005-2760(94)90067-1

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  70. Purification and Properties of phospholipase D from Streptomyces antibioticus 査読有り

    Kikuo Shimbo, Yugo Iwasaki, Tsuneo Yamane and Kazuo Ina

    Biosci. Biotechnol. Biochem.   57 巻 ( 11 ) 頁: 1946-1948   1993年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

  71. Chapter 4, Enzymatic Modification of Phospholipids by Phospholipase D 招待有り 査読有り

    Damnjanović J, Yugo Iwasaki Y

    "Lipid Modification by Enzymes and Engineered Microbes" (Edited by U.T. Bornscheuer), AOCS press,   4 巻   頁: 69 - 88   2018年

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    担当区分:責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1016/B978-0-12-813167-1.00004-9

  72. Salt-induced increase in the yield of enzymatically synthesized phosphatidylinositol and the underlying mechanism 査読有り

    Michiko Muraki, Jasmina Damnjanovic, Hideo Nakano, Yugo Iwasaki

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   122 巻 ( 3 ) 頁: 276 - 282   2016年9月

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    担当区分:責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SOC BIOSCIENCE BIOENGINEERING JAPAN  

    The purpose of this study was to improve the efficiency of enzymatic synthesis of phosphatidylinositol (PI) from phosphatidylcholine (PC) and myo-inositol in a phospholipase D (PLD)-mediated transphosphatidylation. A conventional biphasic reaction system consisting of ethyl acetate and an aqueous buffer afforded PI with a yield of 14 mol%. In contrast, the reaction performed in the presence of high concentration (0.8-4.3 M) of NaCl in the aqueous phase showed improved PI yield in a NaCl concentration-dependent manner. At 4.3 M NaCl, PI yield of as much as 35 mol% was achieved. The increase in the PI yield offered by other tested salts varied; however, we observed that some salts caused inactivation of the enzyme when used at high concentrations. Although NaCl at high concentration increased the apparent hydrolytic activity on aggregated PC, it decreased the activity towards monomeric PC, indicating that high concentration of salt intrinsically inhibits the enzyme. Binding assays revealed that PLD re-localized from the aqueous phase to the solvent-buffer interface, where the enzymatic reaction takes place, in the presence of both, the salt and PC. Hence, we concluded that improvement of the PI synthesis in the presence of salt occurs mainly due to the accumulation of the enzyme at the interface by strengthening the hydrophobic interactions, by which the apparent activation outweighs the salt-induced inhibitory effect. Using this improved system, several PI with defined structures, namely sn-1, 2-dioleoyl-PI, sn-1-palmitoyl-2-oleoyl-PI, and sn-1-stearoyl-2-arachidonoyl-PI, were successfully synthesized with overall yields of 25-37%, and PI isomeric purities of 91-96%. (C) 2016, The Society for Biotechnology, Japan. All rights reserved.

    DOI: 10.1016/j.jbiosc.2016.02.011

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  73. Direct Enzymatic Synthesis of 1-Phosphatidyl-β-D-glucose by Engineered Phospholipase D 査読有り

    Arisa Inoue, Masaatsu Adachi, Jasmina Damnjanović, Hideo Nakano, Yugo Iwasaki

    ChemistrySelect   1 巻 ( 13 ) 頁: 4121 - 4125   2016年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1002/slct.201600839

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  74. Salinity induces membrane structure and lipid changes in maize mesophyll and bundle sheath chloroplasts 査読有り

    Eiji Omoto, Yugo Iwasaki, Hiroshi Miyake, Mitsutaka Taniguchi

    PHYSIOLOGIA PLANTARUM   157 巻 ( 1 ) 頁: 13 - 23   2016年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY  

    The membranes of Zea mays (maize) mesophyll cell (MC) chloroplasts are more vulnerable to salinity stress than are those of bundle sheath cell (BSC) chloroplasts. To clarify the mechanism underlying this difference in salt sensitivity, we monitored changes in the glycerolipid and fatty acid compositions of both types of chloroplast upon exposure to salinity stress. The monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) contents were higher in MC chloroplasts than in BSC chloroplasts, in both the presence and absence of salt treatment. Under salt conditions, the MGDG level in MC chloroplasts was significantly lower than under normal conditions, while it was unchanged in BSC chloroplasts. In both types of chloroplast, the contents of DGDG, phosphatidylglycerol and phosphatidylinositol remained at the same levels in control and salt-treated plants, whereas sulfoquinovosyldiacylglycerol and phosphatidylcholine were significantly lower and higher, respectively, upon salt treatment. In addition, the fatty acid composition and double bond index of individual lipid classes were changed by salt treatment in both BSC and MC chloroplasts, although these factors had no effect on glycerolipid content. These findings suggest that the difference in salt sensitivity of MC and BSC chloroplast membranes is related to differences in MGDG responses to salinity. Thus, we propose that the low MGDG content and the low sensitivity of MGDG to salinity in BSC chloroplasts render them more tolerant than MC chloroplasts to salinity stress.

    DOI: 10.1111/ppl.12404

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  75. Extracellular production of phospholipase A(2) from Streptomyces violaceoruber by recombinant Escherichia coli 査読有り

    Daiki Takemori, Kenta Yoshino, Chisato Eba, Hideo Nakano, Yugo Iwasaki

    PROTEIN EXPRESSION AND PURIFICATION   81 巻 ( 2 ) 頁: 145 - 150   2012年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ACADEMIC PRESS INC ELSEVIER SCIENCE  

    Phospholipase A(2) (PLA(2)) from Streptomyces violaceoruber was successfully produced extracellularly in an active form by using a recombinant strain of Escherichia coli. The PLA(2) gene, which was artificially synthesized with optimized codons for E. coli and fused with pelB signal sequence, was expressed in E. coli using pET system. Most of the enzyme activity was detected in the culture supernatant with negligible activity in the cells. The recombinant enzyme was purified to homogeneity from the culture supernatant simply by ammonium sulfate precipitation and an anion exchange chromatography. The purified enzyme showed a specific activity comparable to that of the authentic enzyme. The recombinant enzyme had the same N-terminal amino acid sequence to that of the mature protein, indicating the correct removal of the signal peptide. An inactive PLA(2) with a mutation at the catalytic center was also secreted to the culture medium, suggesting that the observed secretion was not dependent on enzymatic activity. A simple screening method for the PLA(2)-producing colonies was established by detecting clear zone formation around the colonies on agar media containing lecithin. This is the first example of direct extracellular production of active PLA(2) by recombinant E. coli. (C) 2011 Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.pep.2011.10.002

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  76. Isolation of Phospholipase D Mutants Having Phosphatidylinositol-Synthesizing Activity with Positional Specificity on myo-Inositol 査読有り

    Atsushi Masayama, Kaori Tsukada, Chika Ikeda, Hideo Nakano, Yugo Iwasaki

    CHEMBIOCHEM   10 巻 ( 3 ) 頁: 559 - 564   2009年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY-V C H VERLAG GMBH  

    Phospholipase D (PLD) mutants that have phosphatidylinositol (PI)-synthesizing activity with positional selectivity towards 1- or 3-OH groups of myo-inositol hove been isolated. A mutant PLD library, in which site-directed saturation mutations were introduced in vitro at positions 187, 191, and 385 of the wild-type PLD of Streptomyces antibioticus, was screened for PI-synthesizing mutants. TLC and HPLC analyses of the PI synthesized by the isolated mutant PLDs revealed that three mutants, namely 187D/191Y/385R (DYR), 187A/191Y/385R (AYR), and 787M/191Y/385R (MYR), selectively generated 1- or 3-PI among the other possible PI positional isomers. Taking into account the consensus sequence of the three mutants, a series of mutants, 187X/191Y/385R (XYR), was constructed and analyzed. Almost all the XYR mutants generated 1(3)-PI selectively, thus suggesting that the Y385R mutation contributed to the selectivity for the 1(3)-PI synthesis. The XYR mutants showed similar phosphatidylcholine-hydrolyzing activity among the mutants, but the PI-synthesizing activities were different depending on the amino acid at position 187. In particular, aromatic amino acids at position 187 greatly reduced the PI-synthesizing activity. The ratios of 1-PI versus 3-PI in the PIs synthesized with the XYR mutants were analyzed by selective hydrolysis with PI-specific phospholipase C. It was found that 187H/191Y/385R (HYR) generated I-PI more than 3-PI (ratio = 7.3), whereas 187T/191Y/385R (TYR) generated 1-PI less than 3-PI (ratio = 2:8). This confirmed that the amino acid at position 187 determined the selectivity between 1-PI and 3-PI formation.

    DOI: 10.1002/cbic.200800651

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  77. ホスホリパーゼDの機能改変 招待有り 査読有り

    昌山敦,鈴木淳巨,岩崎雄吾

    生物工学会誌   87 巻   頁: 274-276   2009年

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    担当区分:筆頭著者   記述言語:日本語  

  78. Streptomyces phospholipase D mutants with altered substrate specificity capable of phosphatidylinositol synthesis 査読有り

    Atsushi Masayama, Tetsuya Takahashi, Kaori Tsukada, Seigo Nishikawa, Rie Takahashi, Masaatsu Adach, Kazushi Koga, Atsuo Suzuki, Takashi Yamane, Hideo Nakano, Yugo Iwasaki

    CHEMBIOCHEM   9 巻 ( 6 ) 頁: 974 - 981   2008年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY-V C H VERLAG GMBH  

    The substrate specificity of a phospholipase D (PLD) from Streptomyces antibioticus was altered by site-directed saturation mutagenesis, so that it was able to synthesize phosphatidylinositol (PI). Mutations were introduced in the pId gene at tile positions corresponding to three amino acid residues that might be involved in substrate recognition, and the mutated genes were expressed in Escherichia coli BL21 (DE3). High-throughput screening of approximately 10000 colonies for PI-synthesizing activity identified 25 PI-synthesizing mutant PLDs. One of these mutant enzymes was chosen for further analysis. The structure of the PI synthesized with the mutant enzyme was analyzed by HPLC-MS and NMR. It was found that the mutant enzyme generated a mixture of structural isomers of Pis with the phosphatidyl groups connected at different positions of the inositol ring. The phosphatidylcholine-hydrolyzing activity of the mutant PLO was much lower than that of the wild-type enzyme. The mutant enzyme was able to transphosphatidylate various cyclohexanols with a preference for bulkier compounds. This is the first example of alteration of the substrate specificity of PLO and of PI synthesis by Streptomyces PLO.

    DOI: 10.1002/cbic.200700528

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  79. ホスホリパーゼDによるリン脂質の変換 招待有り

    岩崎雄吾、昌山敦、中野秀雄

    微生物によるものづくり 監修:植田充美 シーエムシー出版     頁: 100-105   2008年

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    担当区分:筆頭著者   記述言語:日本語  

  80. Direct separation of regioisomers and enantiomers of monoacylglycerols by tandem column high-performance liquid chromatography

    Li Deng, Hideo Nakano, Yugo Iwasaki

    JOURNAL OF CHROMATOGRAPHY A   1165 巻 ( 1-2 ) 頁: 93 - 99   2007年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER SCIENCE BV  

    An HPLC-based method for direct separation of the regioisomers and enantiorners of rnonoacylglycerols (MAGs), i.e. sn-I-MAG, sn-2-MAG and sn-3-MAG, has been established. The method employs a tandem column system, in which two different columns (a conventional silica gel column and an enantioselective column) are connected in series. Three isomers of monooleoylglycerols (MOGs) and monolinoleoylglycerols (MLGs) were resolved on the system with resolution factor (R,) of more than 1.1 between adjacent peaks. In addition, all types of oleoylglycerols, i.e. trioleoylglycerol (TOG), sn-1,2-dioleoylglycerol (DOG), sn-2,3-DOG, sn-1,3-DOG, sn-1-MOG, sn-3-MOG and sn-2-MOG, were successfully separated on the tandem column system, although baseline separation of the enantiomers was not achieved. By means of the established analytical method, the reaction course of Candida antarctica lipase B (CALB)-mediated esteritication of glycerol with oleic acid was monitored. It was found that sn-l-MOG and sn-2,3-DOG were preferably generated over sn-3-MOG and sn-1,2-DOG, respectively, in the early stage of the reaction, and the maximal enantiomer excess (%ee) of sn-1-MOG and sn-2,3-DOG were 32 and 53%, respectively, at 2h. The enantiomeric purities of these chiral acylglycerols decreased after prolonged reaction. The mechanisms for the formation of these chiral acylglycerols are discussed. (c) 2007 Elsevier B.V. All rights reserved.

    DOI: 10.1016/j.chroma.2007.07.073

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書籍等出版物 2

  1. 新版 生物反応工学.

    山根 恒夫,中野 秀雄, 加藤 雅士, 岩崎 雄吾, 河原崎 泰昌, 志水 元亨( 担当: 分担執筆)

    産業図書  2016年 

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    記述言語:日本語 著書種別:教科書・概説・概論

  2. 応用微生物学 第3版 (横田篤, 大西康夫, 小川 順 編)

    岩崎雄吾( 担当: 分担執筆 ,  範囲: 「3) 脂質,テルペノイド」(第5章 微生物の代謝),pp.128–131)

    文永堂出版  2016年 

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    記述言語:英語

MISC 1

  1. Synthesis of phosphatidylinositol catalyzed by thermostable variants of Streptomyces phospholipase D

    Jasmina Damnjanovic, Hideo Nakano, Yugo Iwasaki  

    NEW BIOTECHNOLOGY29 巻   頁: S87 - S88   2012年9月

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:ELSEVIER SCIENCE BV  

    DOI: 10.1016/j.nbt.2012.08.245

    Web of Science

講演・口頭発表等 27

  1. Synthesis of phosphatidylinositol using phospholipase D with altered substrate specificity 国際会議

    98th Annual meeting of American Oil Chemists' Society 

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    開催年月日: 2007年5月

    記述言語:英語   会議種別:口頭発表(招待・特別)  

    国名:日本国  

  2. 放線菌ホスホリパーゼDの基質特異性改変

    岩崎雄吾

    第5回 脂質工学研究部会 講演会 

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    開催年月日: 2007年3月

    記述言語:日本語   会議種別:口頭発表(招待・特別)  

    国名:日本国  

  3. Altering substrate specificity of phospholipase D by directed evolution 国際会議

    Japan-Italy Symposium of New Trends in Enzyme Science and Technology 

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    開催年月日: 2006年11月

    記述言語:英語   会議種別:ポスター発表  

    国名:日本国  

  4. Altering substrate specificity of phospholipase D by directed evolution 国際会議

    9th Japan-China-Korea Joint Symposium on Enzyme Engineering 

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    開催年月日: 2006年11月

    記述言語:英語   会議種別:ポスター発表  

    国名:日本国  

  5. Direct separation of regio- and enantiomeric isomers of monoacylglycerols by a tandem column HPLC 国際会議

    9th Japan-China-Korea Joint Symposium on Enzyme Engineering 

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    開催年月日: 2006年11月

    記述言語:英語   会議種別:ポスター発表  

    国名:日本国  

  6. Stereoselective deacylation of triacylglycerol by lipase-catalyzed ethanolysis 国際会議

    World Conference and Exhibition on Oilseed and Vegetable Utilization 

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    開催年月日: 2006年8月

    記述言語:英語   会議種別:口頭発表(招待・特別)  

  7. Altering substrate specificity of Streptomyces phospholipase D by directed evolution approach. 国際会議

    97th Annual meeting of American Oil Chemists' Society 

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    開催年月日: 2006年4月

    記述言語:英語   会議種別:口頭発表(招待・特別)  

    国名:日本国  

  8. ホスファチジルイノシトール合成活性を有する変異型ホスホリパーゼDの創製

    高橋哲也,山根恒夫,中野秀雄,岩崎雄吾

    日本農芸化学会2006年度大会 

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    開催年月日: 2006年3月

    記述言語:日本語   会議種別:口頭発表(一般)  

    国名:日本国  

  9. Production of phosphatidylserine by phospholipase D 国際会議

    72th Annual Meeting of KoSFoST 

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    開催年月日: 2005年6月

    記述言語:英語   会議種別:口頭発表(招待・特別)  

  10. Phospholipase D -mediated synthesis of phosphatidylserine without toxic organic solvent 国際会議

    96th Annual meeting of American Oil Chemists' Society 

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    開催年月日: 2005年5月

    記述言語:英語   会議種別:口頭発表(招待・特別)  

    国名:日本国  

  11. Stereoselective ethanolysis of triacylglycerols by lipases.

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    開催年月日: 2005年3月

    記述言語:英語   会議種別:口頭発表(一般)  

    国名:日本国  

  12. Direct separation of regio- and enantiomeric isomers of diacylglycerols by a tandem column high-performance liquid chromatography.

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    開催年月日: 2004年9月

    記述言語:英語   会議種別:口頭発表(一般)  

    国名:日本国  

  13. Phospholipase Dによるレシチンのphosphatidylserineへの変換

    山根恒夫、岩崎雄吾

    日本生物工学会平成16年度大会 

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    開催年月日: 2004年9月

    記述言語:日本語   会議種別:口頭発表(招待・特別)  

    国名:日本国  

  14. Enzymatic synthgesis of asymmetrical structured lipids 国際会議

    11th European Congress on Biotechnology 

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    開催年月日: 2003年8月

    記述言語:英語   会議種別:ポスター発表  

  15. Biosynthetic pathway of diepoxy bicyclic fatty acids from linoleic acid by Clavibacter sp. ALA2. 国際会議

    2001 Annual Meeting of Society for Industrial Microbiology 

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    開催年月日: 2001年7月

    記述言語:英語   会議種別:ポスター発表  

    "Adam's Mark Hotel St. Louis, MO U.S.A."

  16. Value-added products through biotransformation: Tetrahydrofuranyl fatty acid and sn2-monoacylglycerides. 国際会議

    92 nd Annual meeting of American Oil Chemists' Society 

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    開催年月日: 2001年5月

    記述言語:英語   会議種別:口頭発表(一般)  

  17. EPA含有構造脂質の高純度酵素合成

    山根恒夫,岩崎雄吾,保居守,イリメスク ロクサナ,秦和彦

    日本農芸化学会2000年度大会 

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    開催年月日: 2000年4月

    記述言語:日本語   会議種別:ポスター発表  

    国名:日本国  

  18. 放線菌ホスホリパーゼDの反応機構:ホスファチジル-酵素中間体の検出

    岩崎雄吾,堀池聡子,松島加代子,山根恒夫"\

    日本生物工学会平成11年度大会 

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    開催年月日: 1999年9月

    記述言語:日本語   会議種別:口頭発表(一般)  

    国名:日本国  

  19. "HPLC, GC, TLC/FIDによる脂質の分析~酵素による脂質変換反応を例にして~"

    岩崎雄吾:

    平成11年度日本油化学会若手の会サマースク-ル「機器分析で何がわかるか」 

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    開催年月日: 1999年7月

    記述言語:日本語   会議種別:口頭発表(招待・特別)  

    国名:日本国  

  20. Production of phospholipase D of Streptomyces antibioticus using recombinant Escherichia coli. 国際会議

    90th Annual meeting of American Oil Chemist's Society 

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    開催年月日: 1999年5月

    記述言語:英語   会議種別:ポスター発表  

  21. リパーゼによるシングルセルオイルからの構造脂質の合成.

    岩崎雄吾,山根恒夫

    日本油化学会油化学討論会 

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    開催年月日: 1998年9月

    記述言語:日本語   会議種別:口頭発表(一般)  

    国名:日本国  

  22. リパーゼによる構造脂質の合成

    "岩崎雄吾, Jeong Jun Han, Roxana Rosu , 山根恒夫"

    平成10年度日本生物工学会大会シンポジウム 「脂質工学の最近のトピックス」 

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    開催年月日: 1998年9月

    記述言語:日本語   会議種別:口頭発表(招待・特別)  

    国名:日本国  

  23. リパーゼを用いるアシドリシス反応によるシングルセルオイルからの構造脂質の合成

    岩崎雄吾,成田美保,山根恒夫

    日本農芸化学会1998年度大会 

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    開催年月日: 1998年4月

    記述言語:日本語  

    国名:日本国  

  24. 放線菌ホスホリパーゼDの組換え大腸菌による大量生産と結晶化

    岩崎雄吾,水本耕作,福地英里子,山根恒夫,鈴木淳巨,山根隆

    日本農芸化学会中部支部第120回例会 

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    開催年月日: 1997年10月

    記述言語:日本語  

    国名:日本国  

  25. オリーブ油のグリセロリ シス反応における固定化リパーゼの再利用性

    "岩崎雄吾,ロクサナ・ロシュ,宇於崎友紀,山根恒夫"

    日本油化学会油化学討論会 

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    開催年月日: 1996年11月

    記述言語:日本語  

    国名:日本国  

  26. 放線菌Streptomyces antibioticus の生産するホスファチジルイノシトール特異的ホスホリパーゼC遺伝子のクローニング

    "岩崎雄吾,市橋亜紀子,中野秀雄,山根恒夫"

    日本農芸化学会1996年度大会 

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    開催年月日: 1996年4月

    記述言語:日本語  

    国名:日本国  

  27. 放線菌 Streptomyces antibioticusの生産するホスファチジルイノシトール特異的ホスホリパーゼCの精製と性質

    "岩崎雄吾,山根恒夫:"

    第34回油化学討論会 

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    開催年月日: 1995年10月

    記述言語:日本語  

    国名:日本国  

▼全件表示

共同研究・競争的資金等の研究課題 6

  1. 酵素の耐熱安定化のためのループトリミング法の確立とホスホリパーゼDへの応用

    2012年4月 - 2014年3月

    旭硝子財団研究助成 

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    資金種別:競争的資金

  2. ホスファチジルイノシトール合成型ホスホリパーゼDの位置特異性の向上

    2011年4月 - 2012年3月

    日本応用酵素協会研究助成 

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    資金種別:競争的資金

  3. 酵素によるイノシトールリン脂質およびイノシトールリン酸の合成

    2006年8月 - 2009年3月

    生研センター新技術・新分野創出のための基礎研究推進事業 

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    資金種別:競争的資金

  4. 機能性リン脂質の省エネ合成法の開発

    2005年 - 2006年

    近畿経済産業局、地域新生コンソーシアム研究開発事業 

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    資金種別:競争的資金

  5. 酵素による構造脂質の不斉合成

    2002年4月 - 2003年3月

    長瀬科学技術振興財団研究助成金 

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    資金種別:競争的資金

  6. 酵素による高度不飽和脂肪酸誘導体合成のための工学

    1995年1月

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    受託研究費

▼全件表示

科研費 12

  1. 情動制御作用を有するプラズマローゲンのデザインと脳内輸送経路および標的細胞の解明

    研究課題/研究課題番号:20K07918  2020年4月 - 2023年3月

    宇田川 潤

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    担当区分:研究分担者  資金種別:競争的資金

    脳内のエタノールアミンプラズマローゲン(PlsEtn)は哺乳動物の行動に関係すると考えられている。本研究では、発達障がいや精神疾患の予防や治療に応用可能な高機能PlsEtnの創出を目指し、以下の実験を行う。
    ①ビニールエーテル結合や多価不飽和脂肪酸の有無など、分子構造の異なるリン脂質を合成し、ラットに投与して行動変化を観察する。これにより分子構造と機能との関連を明らかにできる。
    ②安定同位体標識PlsEtnを合成し、脳内移行経路や脳内動態を調べる。これにより高機能PlsEtnの標的となる細胞や反応系が明らかとなる。
    ③これらの知見をもとに、より高機能なPlsEtnをデザインする。

  2. 人工リン脂質を利用した直交型リポソーム融合法の開発とその応用

    研究課題/研究課題番号:19K05160  2019年4月 - 2022年3月

    岩崎 雄吾

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    担当区分:研究代表者  資金種別:競争的資金

    配分額:4420000円 ( 直接経費:3400000円 、 間接経費:1020000円 )

    脂質小胞の膜融合はリポソーム工学において重要な要素技術であるが、従来法には直交性がなく、目的とする異種小胞間での融合を選択的に達成することが困難であった。
    本課題では、極性頭部を修飾した各種人工リン脂質を合成して膜小胞に埋め込み、それらの相互作用を利用して直交型異種間膜融合系を開発する。さらに、鋳型DNA封入リポソームと無細胞タンパク合成試薬封入リポソームを試験管内で直交融合させてタンパク合成を行う。本法によりリポソームへの選択的な物資供給が可能になり、膜タンパク質のリポソーム内分子進化工学などへの応用が期待できる。
    細胞間やリポソーム間の融合は、細胞工学やリポソーム工学において重要な要素技術である。一般に同種小胞間の融合は異種間融合よりも起こりやすいが、異種小胞間での融合を人工リン脂質を用いて誘導できれば有用である。本課題では1.極性頭部に修飾を施した人工リン脂質を合成し、それを含有するリポソームにおいてリン脂質間の共有結合による直交型融合、2リポソーム上にタンパク質を結合させ、タンパクータンパク相互作用を利用したリポソーム融合を試み、異種間直交型融合を確立を目的とする。
    <BR>
    極性頭部にアジド基あるいはアルキン基を導入した人工リン脂質を合成した。2種の人工リン脂質は緩衝液中でクリック反応が進行することが確認された。次に、アジド型、アルキン型それぞれの人工リン脂質を含有するリポソームを調製し、両者を混合してクリック反応を行った。しかしリポソーム融合を確認するには至らなかった。
    <BR>
    リポソーム表面へのタンパク質結合のため、極性部にチオエステル基あるいはクロロアルカン基を結合させた人工リン脂質を合成した。チオエステル型リン脂質を含有するリポソームを調製し、N末端にシステインを持つモデルタンパクとN-to-S転移反応を行った。プロテナーゼKによる分解物をLC-MSで分析したところ、システインが結合した脂質を検出された。同様にクロロアルカン型リン脂質含有リポソームとハロタグタンパクとの結合実験も試みているが結合を確認するまでには至らなかった。
    1. リン脂質間の直接結合によるリポソーム融合
    極性頭部にアジド基あるいはアルキン基を導入した人工リン脂質を酵素反応と化学反応を併用して合成できた。2種の人工リン脂質を緩衝液中で混合し銅存在下で環化付加反応を行った。LC-MS解析により予想された反応生成物を検出され、2種のリン脂質は直交的に反応することが確認できた。次に、アジド型、アルキン型それぞれの人工リン脂質を含有するリポソームを調製した。それぞれのリポソームを識別するためにGFPおよびRFPを内封した。両者を混合してクリック反応を行い、蛍光顕微鏡およびFACSで分析したが、リポソーム融合が起きたと判断できる結果は得られなかった。
    <BR>
    2タンパク質結合型リン脂質を介したリポソーム融合
    リポソーム表面へのタンパク質結合のため、極性部にチオエステル基あるいはクロロアルカン基を結合させた人工リン脂質を合成した。チオエステル型リン脂質を含有するリポソームを調製し、モデルタンパクとしてN末端にシステインを持つGFP(N-Cys-GFP)を混合し、メルカプトフェニル酢酸存在下でN-to-S転移反応を行った。反応後,プロテナーゼK処理によりタンパク質部分を分解し、LC-MSでリン脂質脂質を分析したところ、システインが結合した脂質を検出できた。このことから、N-Cys-GFPとチオエステルリン脂質が意図した通りに共有結合したことが推定された。同様にクロロアルカン型リン脂質含有リポソームとハロタグ融合RFPとの結合実験も試みているが結合を確認するまでには至っていない。
    1. リン脂質間の直接結合によるリポソーム融合
    融合が確認できなかった原因として、リポソーム表面からのアジド基およびアルキン基のが近接しすぎており、リン脂質の負電荷の反発のために両リポソームが接近できない可能性がある。このため、極性頭部と反応性官能基の間にリンカー構造を挿入した新規な人工リン脂質を合成し、融合を試みる。
    <BR>
    2 タンパク質結合型リン脂質を介したリポソーム融合
    ここではタンパク質の構造を損なうことなくリポソーム表面に結合させることが第一の目的である。チオエステルリン脂質との結合は期待通りに進行したと思われるので、顕微鏡観察かFACS分析により、リポソーム上に結合していることを確定させる。一方、ハロタグタンパクとの結合を検証するため、極性部にハロアルカン、脂肪酸残基に蛍光色素を結合したリン脂質を合成し、ハロタグタンパクが蛍光リン脂質により標識されるかを検証する。

  3. 数μLの血液中の脂肪酸を存在状態別に迅速定量するマルチステップ反応熱分解法の開発

    研究課題/研究課題番号:18K05186  2018年4月 - 2021年3月

    石田 康行

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    担当区分:研究分担者  資金種別:競争的資金

    本年度は、昨年度に構築した2段階式の反応熱分解法を、血清中の多価不飽和脂肪酸(PUFA)の分析に適用することを当初の計画としていた。しかし、各種の脂質標準試料を用いて反応熱分解条件の再検討を行った際に、反応性に関わる大きな問題に直面した。その問題とは、初年度に開発した化学反応場は、一部のPUFA成分については加水分解およびメチル化をまったく引き起こせないということであった。具体的には、遊離型、トリグリセリド型およびリン脂質型のPUFA成分については反応熱分解を誘起できるが、コレステリルエステル型については全く反応を引き起こせないことが判明した。この問題を解決できなければ、当該申請課題の目標実現は極めて困難である。
    そこで、研究計画を若干変更して、コレステリルエステルについても作用する化学反応場の構築に比較的多くの時間を充てることとなった。具体的には、1) 様々な化学試薬の探求、2)反応温度や試薬の導入量などの反応条件至適化、および3) キャリアーガス流量や注入口条件などのGC関連条件の最適化を行った。その結果、反応試薬として高い誘起効果と共鳴効果を併せ持つアンモニウム塩型の有機アルカリを選択し、温度および導入量をそれぞれ200℃および2400倍(試料成分に対する試薬の物質量比)に設定したところ、コレステリルエステル中の脂肪酸成分を50%以上の回収率でもって分析できることを見出した。
    こうして新たに構築した化学反応場とGCを連結した結果、わずか1マイクロリットルの血清に含まれるEPAやDHAをはっきりと検出することに成功した。さらに、それらのピーク面積を基にして、含有量の定量も高感度に行うことができた。以上の経緯から、当初の研究計画から若干遅れることにはなったものの、血清中のPUFAを高感度に定量する化学反応場を構築することができた。
    今年度、実際の血清に含まれる多価不飽和脂肪酸(PUFA)の高感度検出を試みたところ、反応性に関する大きな問題に直面した。各種のPUFA標準試料を用いて、反応効率を確認した結果、遊離やトリグリセリド型のPUFAは高効率に加水分解とメチル化を誘起できたが、コレステロールに結合した当該成分についてはほとんど反応を引き起こせないことが判明した。予想外の問題であるとともに、本研究の目標達成のためには解決が欠かせない課題である。そこで実験計画を若干変更し、コレステリルエステルについても高い反応性を示す反応場の構築を行った。その結果、50%以上の反応性を実現することができ、これにより当初の実験目標を達成できる可能性が十分高まった。上記の追加実験により、進捗状況はやや遅れることとなったが、反応性の問題は解決することができた。次年度には計画通りに実験を行える見通しである。
    まず、本手法により得られた測定値の信頼性評価が必要である。測定値の再現性を検証し、精度の観点からの評価を行う。さらに、溶媒抽出やオフラインでの化学反応を利用した、従来法によるクロスチェックも行う。両手法による結果を各種統計的手法を活用してデータ解析することにより、本手法の確度についても評価を試みる。
    さらに、本研究の特徴でもある、PUFA成分の存在状態ごとの分析にも着手する。すでに初年度に構築した2段階反応熱分解法(2種類の異なる化学試薬を血清試料に対して連続的に作用させる方法)を発展的に応用して、遊離型およびエステル型のPUFA成分を個別に定量することを目指す。さらに、酵素反応を化学反応場の前段階に活用することにより、脂質種ごとにPUFA成分を分析することも試みる。

  4. ホスファチジルイノシトールの精密高純度合成のための高機能改変酵素の開発

    研究課題/研究課題番号:25420830  2013年4月 - 2016年3月

    岩崎 雄吾

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    担当区分:研究代表者  資金種別:競争的資金

    配分額:5070000円 ( 直接経費:3900000円 、 間接経費:1170000円 )

    機能性リン脂質ホスファチジルイノシトール(PI)の高純度合成を目的とし、リン脂質変換酵素であるホスホリパーゼD(PLD)の蛋白工学的改変を行った。基質ポケットに変異を導入した変異酵素から、PIを位置選択的に与える変異酵素を同定した。また,PLDの不安定ループを除去することで、安定かつ高選択性を併せ持ったPLDの作出にも成功した。さらにPIの収率を高める為に酵素反応条件を詳細に検討し、反応系に高濃度のNaClの添加という極めて単純な方法でPI収率を飛躍的に高める事に成功した。さらに、反応後の副生物であるホスファチジン酸を分解する酵素を探索し、適した酵素を産生する微生物を同定した。

  5. 無細胞系ギガスクリーニング法の開発と応用

    研究課題/研究課題番号:23360367  2011年4月 - 2015年3月

    中野 秀雄

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    担当区分:研究分担者  資金種別:競争的資金

    蛋白質・核酸などの機能分子創製技術は、生命科学・工学、あるいは医療等の産業において、最重要な研究課題のひとつであるものの、これらは精密な分子設計が不可能であるため、膨大なライブラリーを構築し、機能によるスクリーニングする系の開発が欠かせない。本研究では、容積1pL程度の微小液滴中で様々な生化学・分子生物学的反応が可能な系を構築し、酸化還元酵素や、トランスグルタミナーゼ、RNAポリメラーゼなどの酵素を対象として、ギガ(10の9乗)スケールの分子スクリーニングが可能なシステムを構築した。さらに本システムの効率向上を目的として、均一にかつ高速に水/油エマルジョンを作製できる装置も合わせて開発した。

  6. 改変型ホスホリパーゼDによるホスファチジルイノシトールの位置異性体選択的合成

    研究課題/研究課題番号:22560770  2010年 - 2012年

    岩崎 雄吾

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    担当区分:研究代表者  資金種別:競争的資金

    配分額:4420000円 ( 直接経費:3400000円 、 間接経費:1020000円 )

    ホスファチジルイノシトール合成型ホスホリパーゼ Dのを高機能化を試みた.本酵素の 1-PI選択性を高めるため基質結合部位に変異を導入したところ,選択性を向上させることに成功した.次に本酵素の耐熱化のため揺らぎの大きな残基ランダム化したライブラリーからスクリーニングを行い,耐熱性が向上した変異体を獲得した.不安定性ループを除去した変異体を作製したところ,耐熱性はさらに向上した。

  7. 無細胞系分子ディスプレイシステムの構築と応用

    研究課題/研究課題番号:19360373  2007年 - 2010年

    中野 秀雄

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    担当区分:研究分担者  資金種別:競争的資金

    DNAをマイクロビーズ上にエマルジョンPCRを用いて提示し、さらにそれがコードするペプチドおよび蛋白質を同じビーズ上に提示させる、新規な無細胞系分子ディスプレイシステムを開発した。この手法はフローサイトメーターを用いた高速なスクリーニングに適応可能である。例としてペプチドの進化工学、酵素のハイスループットスクリーニング、転写因子結合配列の解析に応用し、その有効性を実証した。

  8. イメージング質量分析を用いた酵素のハイスループットスクリーニング法

    研究課題/研究課題番号:17760623  2005年 - 2006年

    岩崎 雄吾

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    担当区分:研究代表者  資金種別:競争的資金

    配分額:3400000円 ( 直接経費:3400000円 )

    本研究では、飛行時間型二次イオン質量分析計(TOF-SIMS)による質量分析イメージングを利用して酵素のハイスループットスクリーニング法の開発を目的としている。
    前年度に引き続き、Candida antarctica由来リパーゼB(CALB)をモデル酵素として、マイクロチャンバー上でパラニトロフェニルパルミテートの加水分解反応を行い、その後TOF-SIMSによる検出・イメージングを試みた。
    CALBをシリカゲルビーズに吸着し、固定化酵素を調製した。表面が平らなシリコンウエーハー上に基質であるパラニトロフェニルパルミテートを含有するアガロースフィルムを作成し、その表面に固定化リパーゼを散布し、37度で放置して酵素反応を行った後、TOF-SIMSを用いて反応生成物であるパラニトロフェノールおよびパルミチン酸の検出を行った。TOF-SIMSの性質上、固定化酵素粒子が存在する部分からは固定化酵素そのものによる物理的な障害によりスパッタリングが起こらず、シグナルが検出できなかった。
    そこで、固定化酵素を磁性シリカゲルビーズを用いて再度調製し、これを磁石を用いて基質マトリックスの最表層部に包埋させる技術を開発した。この技術を利用して、再度リパーゼの検出を行ったところ、非常にクリアに酵素活性を検出することに成功し、TOF-SIMSによる酵素の検出が可能であることを実証することが出来た。
    今後は、TOF-SIMS上のイメージと顕微鏡観察におけるイメージの位置を合わせる方法を解決し、TOF-SIMSにより活性が確認されたリパーゼを、顕微鏡下マイクロマニピュレーションにより回収する方法試みる予定である。この方法が完成すれば酵素の分子進化工学におけるスクリーニングのための非常に強力なツールとなることが期待される。

  9. 酵素の光学選択性に関与する基質認識部位の網羅的解析

    研究課題/研究課題番号:16360411  2004年 - 2006年

    中野 秀雄

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    担当区分:研究分担者  資金種別:競争的資金

    本研究では、安定性に優れ、工業的に有用な微生物リパーゼを構造の枠組みとして用い、その基質結合部位だけに組み合わせ変異を導入し、光学異性体基質によりスクリーニングすることで、様々な基質特異性(光学選択性)、反応性を有するリパーゼ群を創製し、基質認識部位の構造と反応性について網羅的に解析することを目的としている。以下に成果の概要を示す。
    1.Burkholderia cepacia KWI-56リパーゼの基質(3-phenylbutyric ester)に対する光学選択性の反転を目標として、基質-酵素複合体のコンピュータモデルを基に、基質結合ポケットの中にある4アミノ酸残基を標的とした、コンビナトリアルライブラリーをSIMPLEX法により作製した。その一次スクリーニングで得られたデータをファジーニューラルネットワーク(FNN)により解析し,基質の光学選択性とアミノ酸変異に関して一定のルールを導きだした。さらに構築したFNNルールに従い、新たな変異体を作製し、得られたルールが妥当であることを証明した。
    2.Burkholderia cepacia KWI-56リパーゼは、2-phenylbutyrateに対して低い光学選択性しか示さない。そこで本酵素にこの基質に対する光学選択性を付与することを目的に、酵素と反応中間体の構造モデルをコンピュータ上で構築し,基質選択性に関与している可能性があるアミノ酸残基を、疎水ポケット中から4カ所予測し選び出し、疎水性アミノ酸(7種)に限定したコンビナトリアル変位を導入した。SIMPLEX法によりライブラリーを作製し、スクリーニングしたところ、野生体より高い光学選択性を有する変異体を取得することに成功した。
    3.SIMPLEX法をより効率化するため,エマルジョン中でDNTA一分子をビーズ上に固定増幅させる技術を開発した。さらにこれをSIMPLEX法と組み合わせ、エマルジョンPCRによりDNA一分子からライブラリーを作製し、その後384穴プレートによる一ビーズPCRを行い、プレート上にDNAライブラリーを構築した。次に無細胞タンパク質合成系により2.で行ったリパーゼの変異ライブラリーを構築し、上記基質によりスクリーニングを行い、野生型より約20倍活性の上昇した変異体を得ることに成功した。

  10. 連続複数塩基ランダム置換法の開発とホスホリパーゼDの機能改変への応用

    2003年 - 2004年

    科学研究費補助金  若手研究(B),課題番号:15760586

    岩崎 雄吾

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    担当区分:研究代表者 

  11. 連続複数塩基ランダム置換法の開発とホスホリパーゼDの機能改変への応用

    研究課題/研究課題番号:15760586  2003年 - 2004年

    岩崎 雄吾

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    担当区分:研究代表者  資金種別:競争的資金

    配分額:3500000円 ( 直接経費:3500000円 )

    連続複数塩基ランダム置換法(RARE-COMBI法)の検討
    クロラムフェニコールアセチルトランスフェラーゼ遺伝子をモデル鋳型とし、3'末端近傍にミスマッチを有する合成DNAをプライマーとした相補鎖合成をPCR法で検討したところ、(1)DNAポリメラーゼは校正活性の低いものを用いる、(2)反応サイクル数を通常のPCRより長く(50サイクル)設定する、(3)25サイクル終了後にポリメラーゼを再添加する。という三つの条件を満足すれば、ミスマッチプライマーでも通常のプライマーと同様の増幅率が得られ、かつ意図したとおりの変異が導入できることを確認した。また、ミスマッチプライマーとミスマッチの無い完全アニール型プライマーを混合して競合的にPCRを行っても、そのプライマーの投入比の通りに変異DNAが出現することを確認した。
    次に、クラゲ緑色蛍光タンパク質(GFP)をターゲットとして、RARE-COMBI法により特定の制限酵素認識部位を導入するというモデル実験を行った。その結果、予想した通りの変異導入が原理的に可能であることを実証できた。しかしながら、変異導入効率は5%程度と低いため、更なる改良が必要である。
    ホスホリパーゼD(PLD)の機能改変
    RARE-COMBI法が未完成であったため、既存の方法によりPLD遺伝子のコンビナトリアルライブラリを構築した。また、人工リン脂質であるホスファチジルナフトール類を合成基質とした簡便なハイスループット検出法を確立した。この検出法を用いて野生型酵素では本来活性を示さない人工リン脂質であるホスファチジル-1-ナフトールに対してスクリーニングを行ったところ、それを分解する能力を獲得した変異型PLDを取得することに成功した。

  12. 高度不飽和脂肪酸含有構造燐脂質の酵素合成と機能評価

    研究課題/研究課題番号:15360438  2003年 - 2004年

    山根 恒夫

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    担当区分:研究分担者  資金種別:競争的資金

    近年、天然リン脂質の1つであるホスファチジルセリン(PS)やドコサヘキサエン酸(DHA)のような高度不飽和脂肪酸が、脳・中枢神経系に対して有用作用を持つことが報告され、生理機能を持つ機能性食品及び医薬品素材として注目されている。そこで、本研究では構造リン脂質としてDHAを構成脂肪酸として含有するPS(DHA-PS)をターゲット分子とし、培養脳神経細胞における機能評価を行う事を目的とした。天然リン脂質として入手が容易なホスファチジルコリン(PC)とDHAからDHA-PSなど各種の構造リン脂質を合成した。得られた脂質をラット胎児脳から調製した初代培養神経細胞に添加し、その影響を細胞死に伴って細胞から放出される安定な酵素であるLactose dehydrogenase(LDH)活性を用いて評価した。また、Amyloid βタンパク質(Aβ)というアルツハイマー病脳細胞において顕著な蓄積が確認され、神経細胞死を引き起こす蛋白質を加えた培養系に各種脂質を添加し、細胞保護作用の有無をLDH活性で評価した。実験に使用した各種脂質には培養条件により生ずる自然細胞死を抑制する作用は確認出来なかった。しかしながら、1,2-didocosahexaenoyl-phosphatidylcholine,1,2-didocosahexaenoyl-phosphatldylserine,1-oleoyl-2-docosahexaenoyl-phosphatidylserineのAβ毒性に対する神経細胞保護作用が確認され、これら構造リン脂質の有効性が明らかとなった。

▼全件表示

産業財産権 6

  1. リン脂質の塩基交換方法

    山根恒 夫、岩崎雄吾、水元夕希子、笠井正章、岡田孝宏

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    出願人:日清オイリオグループ株式会社

    出願番号:特願2001-301656  出願日:2001年9月

    公開番号:特開2002-272493 

    特許番号/登録番号:3697189  登録日:2005年7月 

    出願国:国内  

  2. 高度不飽和脂肪酸を含有する構造油脂の製造方法

    山根恒夫、岩崎雄吾、ロシュロクサナ、清水延寿、沖田裕司

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    出願人:日本水産株式会社

    出願番号:特願平11-084275  出願日:1999年3月

    公開番号:特開2000-270885 

    出願国:国内  

  3. 新規な油脂組成物の製造方法および用途

    山根恒夫、岩崎雄吾、成田美保

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    出願人:ナガセケムテックス株式会社 、サントリー株式会社

    出願番号:特願平10-242330  出願日:1998年8月

    公開番号:特開2000-60587 

    出願国:国内  

  4. 水和性を高めた高度不飽和脂肪酸含有油脂組成物

    山根恒夫、岩崎雄吾、ロクサナ・ロシュ、土居崎信滋、清水延寿

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    出願人:日本水産株式会社

    出願番号:特願平09-089909  出願日:1997年3月

    公開番号:特開平10-265795 

    特許番号/登録番号:3861941  登録日:2006年10月 

    出願国:国内  

  5. グリセリン脂肪酸エステルの製造法

    南部宏暢、山根恒夫、岩崎雄吾、宇於崎 友紀

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    出願人:太陽化学株式会社

    出願番号:特願平8-103482  出願日:1996年3月

    公開番号:特開平9-268299 

    特許番号/登録番号:3510422  登録日:2004年1月 

    出願国:国内  

  6. グリセリン脂肪酸エステルの製造法

    南部宏暢, 山根恒夫, 岩崎雄吾, 宇於崎友紀

     詳細を見る

    特許番号/登録番号:特許3510422  発行日:2004年1月

▼全件表示

 

担当経験のある科目 (本学) 56

  1. 基礎セミナーB

    2020

  2. 遺伝子工学

    2019

  3. 基礎セミナーB

    2015

  4. バイオテクノロジー(分担)

    2015

  5. Agricultural Sciences (分担)

    2015

  6. 遺伝子工学(分担)

    2015

  7. 基礎セミナーB

    2014

  8. バイオテクノロジー(分担)

    2014

  9. Agricultural Sciences (分担)

    2014

  10. 遺伝子工学(分担)

    2014

  11. 遺伝子工学(分担)

    2013

  12. Agricultural Sciences (分担)

    2013

  13. バイオテクノロジー(分担)

    2013

  14. 基礎セミナーB

    2013

  15. 遺伝子工学(分担)

    2012

  16. 基礎セミナーB

    2012

  17. バイオテクノロジー(分担)

    2012

  18. Agricultural Sciences (分担)

    2012

  19. 基礎セミナーB

    2011

  20. Agricultural Sciences (分担)

    2011

  21. バイオテクノロジー(分担)

    2011

  22. 遺伝子工学(分担)

    2011

  23. 遺伝子工学(分担)

    2010

  24. 基礎セミナーB

    2010

  25. Agricultural Sciences (分担)

    2010

  26. バイオテクノロジー(分担)

    2010

  27. 基礎セミナーB

    2009

  28. 応用生命科学科学生実験(分担)

    2009

  29. 遺伝子工学(分担)

    2009

  30. Agricultural Sciences (分担)

    2009

  31. バイオテクノロジー(分担)

    2009

  32. 基礎セミナーB

    2008

  33. 生命技術科学特論IV(分担)

    2008

  34. Agricultural Sciences (分担)

    2008

  35. 遺伝子工学(分担)

    2008

  36. バイオテクノロジー(分担)

    2008

  37. 応用生命科学科学生実験(分担)

    2008

  38. 生物工学I

    2007

  39. 基礎セミナーB

    2007

  40. バイオテクノロジー

    2007

  41. バイオテクノロジー

    2006

  42. 基礎セミナーB

    2006

  43. 生物工学I

    2006

  44. 基礎セミナーB

    2006

  45. 生命農学概論

    2005

  46. バイオテクノロジー

    2005

  47. バイオテクノロジー

    2005

  48. 生命農学概論

    2005

  49. 生命農学概論

    2004

  50. バイオテクノロジー

    2004

  51. バイオテクノロジー

    2004

  52. 生命農学概論

    2004

  53. 生命農学概論

    2003

  54. バイオテクノロジー

    2003

  55. バイオテクノロジー

    2003

  56. 生命農学概論

    2003

▼全件表示

担当経験のある科目 (本学以外) 2

  1. 環境微生物工学

    2020年10月 - 現在 岐阜大学)

     詳細を見る

    科目区分:学部専門科目 

  2. バイオテクノロジー

    2016年4月 - 現在 中部大学)