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Biochemical characterization and substrate profiling of a new NADH-dependent enoate reductase from Lactobacillus casei

Paper ID Volume ID Publish Year Pages File Format Full-Text
17314 42659 2012 9 PDF Available
Title
Biochemical characterization and substrate profiling of a new NADH-dependent enoate reductase from Lactobacillus casei
Abstract

Carbon–carbon double bond of α,β-unsaturated carbonyl compounds can be reduced by enoate reductase (ER), which is an important reaction in fine chemical synthesis. A putative enoate reductase gene from Lactobacillus casei str. Zhang was cloned into pET-21a(+) and expressed in Escherichia coli BL21 (DE3) host cells. The encoded enzyme (LacER) was purified by ammonium sulfate precipitation and treatment in an acidic buffer. This enzyme was identified as a NADH-dependent enoate reductase, which had a Km of 0.034 ± 0.006 mM and kcat of (3.2 ± 0.2) × 103 s−1 toward NADH using 2-cyclohexen-1-one as the substrate. Its Km and kcat toward substrate 2-cyclohexen-1-one were 1.94 ± 0.04 mM and (8.4 ± 0.2) × 103 s−1, respectively. The enzyme showed a maximum activity at pH 8.0–9.0. The optimum temperature of the enzyme was 50–55 °C, and LacER was relatively stable below 60 °C. The enzyme was active toward aliphatic alkenyl aldehyde, ketones and some cyclic anhydrides. Substituted groups of cyclic α,β-unsaturated ketones and its ring size have positive or negative effects on activity. (R)-(−)-Carvone was reduced to (2R,5R)-dihydrocarvone with 99% conversion and 98% (diasteromeric excess: de) stereoselectivity, indicating a high synthetic potential of LacER in asymmetric synthesis.

► A new NADH-dependent enoate reductase (LacER) was characterized from L. casei. ► LacER displayed an optimum temperature at 50–55 °C and an optimum pH of 8.0–9.0. ► It was active for aliphatic α,β-unsaturated aldehydes, ketones and cyclic anhydrides. ► (R)-(−)-Carvone was reduced to (2R,5R)-dihydrocarvone with 99% conversion and 98% de.

Keywords
Carbon–carbon double bond; Enoate reductase; Bioreduction; Biocatalysis
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Biochemical characterization and substrate profiling of a new NADH-dependent enoate reductase from Lactobacillus casei
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 51, Issue 1, 10 June 2012, Pages 26–34
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