Abstract
An NADPH-dependent carbonyl reductase (PsCR) gene from Pichia stipitis was cloned. It contains an open reading frame of 849 bp encoding 283 amino acids whose sequence had less than 60% identity to known reductases that produce ethyl (S)-4-chloro-3-hydroxybutanoates (S-CHBE). When expressed in Escherichia coli, the recombinant PsCR exhibited an activity of 27 U/mg using ethyl 4-chloro-3-oxobutanoate (COBE) as a substrate. Reduction of COBE to (S)-CHBE by transformants in an aqueous mono-phase system for 18 h, gave a molar yield of 94% and an optical purity of the (S)-isomer of more than 99% enantiomeric excess.
Publication types
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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Acetoacetates / metabolism
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Alcohol Oxidoreductases / genetics*
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Alcohol Oxidoreductases / isolation & purification
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Alcohol Oxidoreductases / metabolism*
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Amino Acid Sequence
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Butyrates / metabolism*
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Cloning, Molecular
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Escherichia coli / genetics
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Fungal Proteins / genetics*
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Fungal Proteins / isolation & purification
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Fungal Proteins / metabolism*
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Gene Expression
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Kinetics
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Molecular Sequence Data
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Open Reading Frames
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Oxidation-Reduction
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Pichia / enzymology*
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Pichia / genetics
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Sequence Alignment
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Sequence Homology, Amino Acid
Substances
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Acetoacetates
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Butyrates
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Fungal Proteins
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ethyl 4-chloro-3-hydroxybutanoate
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ethyl 4-chloro-3-oxobutanoate
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Alcohol Oxidoreductases