Identification and characterization of an ene-reductase from Corynebacterium casei.

The asymmetric reduction of α, ß-unsaturated compounds conjugated with electron-withdrawing group by ene-reductases (ERs) is a valuable method for the synthesis of enantiopure chiral compounds. This study introduced an ER from Corynebacterium casei (CcER) which was heterologously expressed in Escherichia coli BL21(DE3), and the purified recombinant CcER was characterized for its biocatalytic properties. CcER exhibited the highest specific activity at 40 °C and pH 6.5, and showcased appreciable stability below 40 °C over a pH range of 6.0-7.0. The enzyme displayed high resistance to methanol. CcER accepted NADH or NADPH as a cofactor and exhibited a broad substrate spectrum towards α, ß-unsaturated compounds. It achieved complete conversion of 2-cyclohexen-1-one and good performance for stereoselective reduction of (R)-carvone (conversion 98 %, diastereoselectivity 96 %). This study highlights the robustness and potential of CcER.

Modulation of the catalytic performance of OYE3 by engineering key residues at the entrance of the catalytic pocket.

The amino acid residues at the entrance of the catalytic pocket may impose steric hindrance on the substrate to enter the active center of the enzyme. Based on the analysis of the three-dimensional structure of the Saccharomyces cerevisiae old yellow enzyme 3 (OYE3), four bulky residues were chosen and mutated to small amino acids. The results showed that mutation of the W116 residue had interesting impacts on the catalytic performance. All four variants became inactive for the reduction of (R)-carvone and (S)-carvone, but inverted the stereoselectivity for the reduction of (E/Z)-citral. The mutation of the F250 residue had a more positive effect on the activity and stereoselectivity. Two variants, F250A and F250S, showed excellent diastereoselectivity and activity for the reduction of (R)-carvone (de > 99%, c > 99%) and increased diastereoselectivity and activity for the reduction of (S)-carvone (de > 96%, c > 80%). One variant of the P295 residue, P295G, displayed excellent diastereoselectivity and activity only for the reduction of (R)-carvone (de > 99%, c > 99%). Mutation of the Y375 residue had a negative impact on the activity of the enzyme. These findings provide some solutions for rational enzyme engineering of OYE3.

Semi-rational protein engineering of a novel ene-reductase from Galdieria sulphuraria for asymmetric reduction of (R)-carvone and ketoisophorone.

Asymmetric reduction of (R)-carvone and ketoisophorone by an engineered ene-reductase from Galdieria sulphuraria (GsOYE) combined with glucose dehydrogenase for NADPH regeneration were studied. A semi-rational protein engineering was used to enhance the activity and selectivity of GsOYE. Upon the sequence alignment and molecular docking results, two amino acid residues at positions 66 and 270 were selected as saturation mutation sites. Finally, a single substitution variant of GsOYE-N270A with complete conversion (100%) and diastereoselectivity (dep >99%) for reduction of (R)-carvone and a double substitution variant GsOYE-Y66P/N270H with improved stereoselectivity for reduction of ketoisophorone were obtained.

The enzymatic resolution of 1-(4-chlorophenyl)ethylamine by Novozym 435 to prepare a novel triazolopyrimidine herbicide.

The kinetic resolution of (R,S)-1-(4-chlorophenyl)ethylamine was accomplished using a commercial lipase from Candida antarctica (Novozym 435). The performance of this lipase was investigated for the enantioselective amidation of (R,S)-1-(4-chlorophenyl)ethylamine, leaving the target product (S)-1-(4-chlorophenyl)ethylamine in its unreacted form. The effects of various types of solvents and an acyl donor, the molar ratio of the substrate to the acyl donor, and the reaction temperature were studied. The optimum reaction conditions were found to result in amidation with methyl 2-tetrahydrofuroate at 40°C in methyl tert-butyl ether, with a substrate/acyl donor molar ratio of 1:2.4. The conversion rate of (R,S)-1-(4-chlorophenyl)ethylamine was 52%, with an enantiomeric excess of 99% towards the unreacted substrate in a reaction time of 22 hours. Finally, using optically pure (S)-1-(4-chlorophenyl)ethylamine as the raw material, the chemical synthesis of (S)-N-(1-(4-chlorphenyl)ethyl)-2-(5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-ylthio)acetamide, a novel triazolopyrimidine herbicide, was achieved, and the total yield and purity were 83.5% and 95.3%, respectively.

Efficient kinetic resolution of (RS)-1-phenylethanol by a mycelium-bound lipase from a wild-type Aspergillus oryzae strain.

A mycelium-bound lipase from Aspergillus oryzae (AOL) exhibited excellent enantioselectivity for kinetic resolution of (RS)-1-phenylethanol ((RS)-1-PE) in organic solvent. The various reaction parameters affecting the conversion and enantioselectivity were studied, including type of acyl donor, solvent, molar ratio, temperature, enzyme amount, and substrate concentration. The optimum reaction conditions were found to be transesterification with vinyl acetate at 30 °C in methyl tert-butyl ether with a vinyl acetate: (RS)-1-PE molar ratio of 1:1 and an enzyme concentration of 60 g/L. At the optimum reaction conditions, the conversion could reach above 46% with >99% enantiomeric excess of the product, (R)-1-phenylethyl acetate, when the substrate concentration was below 1.4 M. The enzyme displayed an excellent enantioselectivity with an E-value of >200 and a strong tolerance for high substrate concentration of up to 1.8 M. Those results indicated that AOL was a promising biocatalyst in the kinetic resolution of (RS)-1-PE.

Kinetic resolution of alpha-lipoic acid via enzymatic differentiation of a remote stereocenter.

Kinetic resolution of alpha-lipoic acid, a case of remote stereocenter discrimination, was accomplished using lipase from Aspergillus oryzae WZ007. Performance of this lipase was investigated for enantioselective esterification of (S)-alpha-lipoic acid, leaving the target product (R)-alpha-lipoic acid in unreacted form. The effects of chain length of alcohol, type of solvent, molar ratio of alcohol:acid, and reaction temperature were studied. The optimum reaction conditions were found to be esterification with n-octanol at 50 degrees C in heptane with an alcohol:acid molar ratio of 5:1. The conversion rate of alpha-lipoic acid was 75.2%, with an enantiomeric excess of 92.5% towards unreacted substrate in a reaction time of 48 h.