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    • 5. 发明公开
    • METHOD OF MODIFYING ENZYME AND OXIDOREDUCTASE VARIANT
    • 改变酶和氧化还原酶变异体的方法
    • EP1416050A1
    • 2004-05-06
    • EP02738904.8
    • 2002-07-02
    • KANEKA CORPORATION
    • NAKAI, TakahisaMORIKAWA, SouichiKIZAKI, NoriyukiYASOHARA, Yoshihiko
    • C12N15/53C12N9/06C12N1/21C12P7/62
    • C12N9/0006C12P7/62
    • An enzyme modifying method for converting the coenzyme-dependency of an oxidoreductase is developed. Using this method, a novel carbonyl reductase mutant capable of utilizing NADH as a coenzyme is provided. It is also intended to provide a process for enzymatically producing an optically active (S)-4-halo-3-hydroxybutyric ester by utilizing the carbonyl reductase mutant.
      A method for modifying an enzyme itself so as to convert the coenzyme-dependency of a carbonyl reductase which asymmetrically reduces a carbonyl compound to produce an optically active alcohol, a carbonyl reductase having such coenzyme dependency as has been converted from NADPH to NADH which is obtained by the above method, a DNA encoding this enzyme mutant, a plasmid carrying this DNA, a transformant obtained by the transformation with this plasmid, and a process for producing an optically active alcohol by using this enzyme mutant and/or this transformant.
    • 开发了用于转化氧化还原酶的辅酶依赖性的酶修饰方法。 使用该方法,提供了能够利用NADH作为辅酶的新型羰基还原酶突变体。 还旨在提供通过利用羰基还原酶突变体酶促生产光学活性(S)-4-卤代-3-羟基丁酸酯的方法。 一种修饰酶本身以转化羰基还原酶的辅酶依赖性的方法,该羰基还原酶不对称地还原羰基化合物以产生光学活性醇,具有从NADPH转化为NADH的这种辅酶依赖性的羰基还原酶,其获得 通过上述方法,通过使用该酶突变体和/或该转化体,编码该酶突变体的DNA,携带该DNA的质粒,通过该质粒转化而获得的转化体以及用于生产光学活性醇的方法。
    • 10. 发明公开
    • NOVEL CARBONYL REDUCTASE, GENE THEREOF AND METHOD OF USING THE SAME
    • 亲爱的哥伦比亚大公司,德累斯顿VERFAHREN ZU DESSEN VERWENDUNG
    • EP1306438A1
    • 2003-05-02
    • EP01956776.7
    • 2001-08-01
    • KANEKA CORPORATION
    • KIZAKI, NoriyukiYASOHARA, YoshihikoHASEGAWA, Junzo
    • C12N15/53C12N9/02C12N1/21C12P17/10
    • C12N9/0008C12N9/0006C12P17/10C12P41/002
    • The present invention provides a novel polypeptide producing (S)-N-benzyl-3-pyrrolidinol, a DNA coding for it and a method of using them.
      A polypeptide having the following physicochemical properties (1) to (5):

      (1) Action: It asymmetrically reduces N-benzyl-3-pyrrolidinone to produce (S)-N-benzyl-3-pyrrolidinol with NADPH as a coenzyme;
      (2) Optimum action pH: 4.5 to 5.5;
      (3) Optimum action temperature: 40°C to 45°C;
      (4) Molecular weight: About 29,000 as determined by gel filtration analysis, about 35,000 as determined by SDS-polyacrylamide gel electrophoresis analysis;
      (5) Inhibitor: It is inhibited by the divalent copper ion.

      Further, a polypeptide having the amino acid sequence shown under SEQ ID NO:1 in the sequence listing; or
         a polypeptide having an amino acid sequence obtainable from the amino acid sequence shown under SEQ ID NO:1 in the sequence listing by substitution, insertion, deletion and/or addition of one or more amino acids and
         having enzyme activity in asymmetrically reducing N-benzyl-3-pyrrolidinone to produce (S)-N-benzyl-3-pyrrolidinol.
    • 本发明提供了产生(S)-N-苄基-3-吡咯烷醇的新型多肽,其编码的DNA及其使用方法。 具有以下物理化学性质的多肽(1)至(5):(1)作用:不对称地还原N-苄基-3-吡咯烷酮以制备(S)-N-苄基-3-吡咯烷醇,其中NADPH作为辅酶; (2)最佳作用pH:4.5〜5.5; (3)最佳作用温度:40〜45℃; (4)分子量:通过凝胶过滤分析确定约29,000,通过SDS-聚丙烯酰胺凝胶电泳分析测定约35,000; (5)抑制剂:被二价铜离子抑制。 此外,具有序列表中SEQ ID NO:1所示的氨基酸序列的多肽; 或具有通过序列表中SEQ ID NO:1所示的氨基酸序列获得的氨基酸序列的多肽,其通过取代,插入,缺失和/或添加一个或多个氨基酸并具有不对称还原N- 苄基-3-吡咯烷酮,得到(S)-N-苄基-3-吡咯烷醇。