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 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 synthetic potential of LacER in asymmetric synthesis.
authors.affiliation: National Engineering Laboratory for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 Xi Qi Dao, Tianjin Airport Economic Area, Tianjin 300308, China.
authors.forename: Xiuzhen
authors.initials: X
authors.lastname: Gao
authors.forename: Jie
authors.initials: J
authors.lastname: Ren
authors.forename: Qiaqing
authors.initials: Q
authors.lastname: Wu
authors.forename: Dunming
authors.initials: D
authors.lastname: Zhu
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007780:Lacticaseibacillus casei / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D009252:NADPH Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013237:Stereoisomerism; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D009243:NAD; D044925:Oxidoreductases Acting on CH-CH Group Donors; D009252:NADPH Dehydrogenase