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1998 Fiscal Year Final Research Report Summary

cDNA cloning of novel aldehyde reductase gene from yeast and its application to chiral alcohol synthesis

Research Project

Project/Area Number 09660091
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field 応用微生物学・応用生物化学
Research InstitutionTottori University

Principal Investigator

KITA Keiko  Tottori University, Dept. of Biotechnology, Associate Professor, 工学部, 助教授 (70234226)

Co-Investigator(Kenkyū-buntansha) KATAOKA Michihiko  Kyoto University, Graduate School of Agriculture, Assistant Professor, 大学院・農学研究科, 助手 (90252494)
SHIMIZU Sakayu  Kyoto University, Graduate School of Agriculture, Professor, 大学院・農学研究科, 教授 (70093250)
Project Period (FY) 1997 – 1998
KeywordsChiral alcohol / Chiral synton / Yeast / Aldehyde reductase / Glucose dehydrogenase / NADPH-binding / cDNA cloning
Research Abstract

New aldehyde reductases (AR), ARII and ARIII, which reduce ethyl 4-chloro-3-oxobutanoate (4-COBE) to ethyl 4-chloro-3-hydroxybutanoate (CHBE), with NADPH as a cofactor, were purified from Sporobolomyces salmonicolor AKU4429. ARII catalyzed the stereospecific reduction of 4-COBE to (S)-CHBE (92.7% enantiomeric excess (e.e.)), in contrast, ARIII reduced 4-COBE to R-CHBE (38.4% e.e.). ARII reduced aliphatic and aromatic aldehydes, and carbonyl compounds such as camphorquinone, but did not accept aldose as a substrate. The enzyme is a monomer protein with a relative molecular mass of 34,000. Its isoelectric point is 5.0. The NH2-terminal amino acid sequence of ARII is different from that of ARI, which catalyzes the stereospecific reduction of 4-COBB to R-CHBE (100% e.e.). We cloned and sequenced the gene encoding an NADPH-dependent ARII from S. salmonicolor AKU4429. The ARII gene comprises 1,032 bp, is interrupted by 4 introns, and encodes a polypeptide of 37,315 Da. The deduced amino acid … More sequence showed significant similarity to that of members of the mammalian 3β-hydroxysteroid dehydrogenase/plant dihydroflavonol 4-reductase superfamily, but not to those of members of the aldo-keto reductase superfamily or to that of ARI previously isolated from the same organism. The ARII protein was overproduced in Escherichia coli about 2,000-fold per g cells, compared to in the original yeast cells. The enzyme expressed in E. coli was purified to homogeneity, and showed the same catalytic properties as ARII purified from S. salmonicolor. To examine the contribution of the dinucleotide-binding motif, GィイD219ィエD2-X-X-GィイD222ィエD2-X-X-AィイD225ィエD2, located in the NH2-terminal region, for ARII catalysis, we exchanged three amino acid residues in the motif, and purified the respective mutant enzymes. The substrate inhibition of the enzyme by 4-COBE was absent in the GィイD219ィエD2 → A and GィイD222ィエD2 → A mutant enzymes. The AィイD225ィエD2 → G mutant enzyme could reduce 4-COBE when NADPH was replaced by an equimolar concentration of NADH. Less

  • Research Products

    (8 results)

All Other

All Publications (8 results)

  • [Publications] 喜多 恵子: "組換え体大腸菌を用いた光学活性アルコールの合成"生物工学会誌. 76(10). 429-432 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Keiko Kita: "Purification and characterization of new aldehyde reductases from Sporobolomyces salmonicolor AKU4429"J.Mol.Catalysis B : Enzymatic. 6. 305-313 (1999)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Keiko Kita: "Cloning,overexpression,and mutagenesis of the Sporobolomyces salmonicolor AKU4429 gene encoding a new aldehyde reductase,which catalyzes the stereoselective reduction of ethyl 4-chloro-3-oxobutanoate to ethyl-(S)-4-chloro-3-hydroxybutanoate"Appl.Environ.Microbiol.. 65(12). 5207-5211 (1999)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Keiko Kita: "Diversity of 4-chloroacetoacetate ethyl ester-reducing enzymes in yeasts and their application to chiral alcohol synthesis"J.Biosci.Bioeng.. 88(6). 591-598 (1999)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] K. Kita: "Chiral alcohol synthesis with Escherichia coli transformant expressing glucose dehydrogenase gene and aldehyde reductase gene"Seibutu kougaku kaishi. 76(10)(in Japanese). 429-432 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] K. Kita: "Purification and characterization of new aldehyde reductases from Sporobolomyces salmonicolor AKU4429"J. Mol. Catalysis B : Enzymatic. 6. 429-432 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] K. Kita: "Cloning, overexpression, and mutagenesis of the Sporobo-lomyces salmonicolor AKU4429 gene encoding a new aldehyde reductase, which catalyzes the stereoselective reduction of ethyl 4-chloro-3-oxobutanoate to ethyl-(S)-4-chloro-3-hydroxybutanoate"Appl. Environ. Microbiol.. 65(12). 5207-5211 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] K. Rita: "Diversity of 4-chloroacetoacetate ethyl ester-reducing enzymes in yeasts and their application to chiral alcohol synthesis"J. Biosci. Bioeng.. 88(6). 591-598 (1999)

    • Description
      「研究成果報告書概要(欧文)」より

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Published: 2001-10-23  

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