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

Carbon-carbon double bond of alpha,beta-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 kcat of (3.2 +/- 0.2) x 10(3) 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) x 10(3) s(-1), respectively. The enzyme showed a maximum activity at pH 8.0-9.0. The optimum temperature of the enzyme was 50-55 degrees C, and LacER was relatively stable below 60 degrees C. The enzyme was active toward aliphatic alkenyl aldehyde, ketones and some cyclic anhydrides. Substituted groups of cyclic alpha,beta-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. (C) 2012 Elsevier Inc. All rights reserved.