Screening Of An Enoate Reductase And Its Application In Asymmetric Synthesis

Enoate reductases are a group of oxidoreductases catalyzing the reduction of?,?-unsaturated carbon-carbon double bonds in the presence of NAD?P?H.The reduction reaction catalyzed by enoate redutase is attracting to synthesize chiral compounds with one or two stereogenic centres.Enoate reductases are widely present in nature,especially yeast and bacteria.Here,an excellent biocatalyst—Bacillus cereus WZY004was screened from the soil samples and then identified.Furthermore,the gene encoding an enoate reductase was successfully cloned and over-expressed in Escherichia coli BL21?DE3?.In addition,enzymatic and catalytic properties of the purified recombinat enzyme were investigated in details.1.Various strains capable of reducing ketoisophronone as substrate were screened and isolated from the soil samples.The strain WZY004was choosed for further characterization due to its high activity and stereoselectivity.Based on morphological,physiological and 16S rDNA characteristics,the isolated strain WZY004 was identified and named as B.cereus WZY004.When glucose was supplemented as co-substrate,both the yield and e.e.value of the reaction were significantly improved.The increase of shaking speed also could improve the conversion rate but reduce the stereoselectivity.The study of substrate spectrum showed that B.cereus WZY004 had a good regioselectivity for trans-2-hexenal and trans-cinnamaldehyde.The conversion and yield for trans-2-hexenal reduction were 97.2%and 86.6%,respectively,while those for trans-cinnamaldehyde reduction were 95.6%and 87.6%,respectively.The strain WZY004 also demonstrated high stereoselectivity for for the reduction of citral and 2-methyl-2-pentene aldehyde,which e.e.values of product were>99.0%and 95.0%,respectively.2 The gene encoding an enoate reductase was amplified,ligated onto the plasmid pEASY-E1 and subsequently over-expressed in E.coli BL21?DE3?.The purified recombinant enzyme was a FAD-dependent protein,and the values of K_m and V_maxax for ketoisophorone reduction were1.83 mM and 1.13 U/mg,respectively.The enzyme used both NADH and NADPH as coenzyme,while the enzyme activity using NADH as coenzyme was 1.5 times higher than that using NADPH as coenzyme.The optimal activity was observed at p H 7.5 and 60°C,whereas the activity was not stable in the case of>50?.The enzyme exhibited no activitytowardketoisophoroneanalogues,e.g.,isophorone,4-hydroxyisophorone and 3-methyl-2-cyclohexen-1-one,suggesting that C=O at the 4-position of ketoisophorone was critical to the enzyme activity.In addition,the enzyme was active toward various straight-chain alkene.Particularly,the relative activities for the reduction of2-methyl-2-pentenal and citral were 144%and 195%,respectively.3.Two enzymatic catalytic systems were developed for asymmetric reduction of ketoisophonone and citral using glucose dehydrogenase?GDH?from Bacillus subtilis to establish the regeneration of coenzyme.The asymmetric reduction of ketoisophorone was performed in a system containing 50mM Tris-HCl?pH 8.5?,0.5U/ml enoate reductase,100mM substrate,400?M NADH,5.0U/ml GDH,and 500mM glucose.After 24h of reaction under the optimized conditions,the yield and e.e.value were85%and 64%,respectively.The asymmetric reduction of citral was performed in a system containing 50mM Tris-HCl?pH 8.5?,1.0U/ml enoate reductase,10mM substrate,400?M NADH,1.2U/ml GDH and100mM glucose.after 2h of reaction,the yield and e.e.value were 25%and>99%,respectively.The enzyme preferred to the reduction of?Z?-citral rather than that of?E?-citral.