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Gene cloning of an NADPH-dependent menadione reductase from Candida macedoniensis, and its application to chiral alcohol production

Paper ID Volume ID Publish Year Pages File Format Full-Text
18696 42735 2006 8 PDF Available
Title
Gene cloning of an NADPH-dependent menadione reductase from Candida macedoniensis, and its application to chiral alcohol production
Abstract

The gene encoding an NADPH-dependent menadione reductase of Candida macedoniensis AKU4588 was cloned and sequenced. A 1035 bp nucleotide fragment (mer) was confirmed to be the gene encoding the enzyme based on the agreement of N-terminal and internal amino acid sequences. The mer encodes 345 amino acid residues, and the deduced amino acid sequence shows high similarity with those of hypothetical proteins from Debaryomyces, Candida and Saccharomyces, and ketoreductase from Zygosaccharomyces. It includes NADPH-binding motif GXXGXXA in its N-terminal region. These findings suggest that the enzyme belongs to the dihydroflavonol-4-reductase superfamily. An expression vector, pETMER, which contains the full length of the mer, was constructed. Escherichia coli cells harboring pETMER exhibits a 127-fold increase in specific menadione-reducing activity under the control of T7 promoter as compared with that of C. macedoniensis.The asymmetric reduction of 4-chloro-3-oxobutanoate ethyl ester to (S)-4-chloro-3-hydroxybutanoate ethyl ester (CHBE) with E. coli cells, in which both the mer and the glucose dehydrogenase gene were co-expressed, as a catalyst was investigated. The (S)-CHBE formed amounted to 1680 mM (281 mg/ml), the molar yield being 92.2%. The optical purity of the product was 91.6% enantiomeric excess for the (S)-isomer. The calculated turnover number of NADP+ added to CHBE formed was 12,900 mol/mol.

Keywords
Chiral alcohol; Asymmetric reduction; Menadione reductase; Carbonyl reductase; Candida macedoniensis; Dihydroflavonol-4-reductase
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Gene cloning of an NADPH-dependent menadione reductase from Candida macedoniensis, and its application to chiral alcohol production
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 38, Issue 7, 2 May 2006, Pages 944–951
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us