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

Enzyme and Microbial Technology
Xiuzhen GaoDunming Zhu

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 K(m) of 0.034 ± 0.006 mM and k(cat) of (3.2 ± 0.2) × 10³ s⁻¹ toward NADH using 2-cyclohexen-1-one as the substrate. Its K(m) and k(cat) 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 syntheti...Continue Reading

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Citations

Jan 3, 2013·Biotechnology and Bioengineering·Yilei FuDirk Weuster-Botz
Mar 13, 2014·Current Opinion in Chemical Biology·Helen S Toogood, Nigel S Scrutton
May 20, 2015·Fungal Biology·Alice RomagnoloGiovanna Cristina Varese
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Feb 14, 2019·Chembiochem : a European Journal of Chemical Biology·Christin PetersRebecca Buller
Mar 20, 2018·World Journal of Microbiology & Biotechnology·Facundo MarconiPaula Rodriguez

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