Gene Mining,Cloning And Expression Of A Carbonyl Reductase From Bacillus Cereus

Chiral alcohol is an important chiral block for synthesis of various chiral drugs,agrochemicals,natural products and certain special materials.Asymmetric reduction of the corresponding prochiral carbonyl compounds with carbonyl reductase is one of the important routes for the chiral alcohols synthesis.So the carbonyl reductase is one of key factors to this process.In this paper,a gene mining strategy was constructed,and a potential carbonyl reductase in protein database was discovered,cloned and expressed.In this study,after analyzing the amino acid conserved sequences of the carbonyl reductase family,a carbonyl reductase(ApCR)from Aeropyrum pernix K1 was selected as a probe enzyme for novel carbonyl reductase mining.Homologous sequence alignment was performed using the protein database with the ApCR amino acid sequence as the probe,and a candidate enzymes library was constructed.After performing amino acid sequence alignments,simulation and comparison of secondary structures and tertiary structure between the candidate enzyme sequence and the probe sequence,a putative carbonyl reductase,BcCR,which is from Bacillus cereus,was obtained and explored.This putative carbonyl reductase has similar secondary and tertiary structures to the ApCR.And the two carbonyl reductases both have the same amino acid conserved sequences for the SDRs family.Therefore,it is speculated that this protein has similar enzymatic properties and functions as ApCR.The BcCR DNA(738bp)was cloned from the genomic DNA of B.cereus by PCR.The plasmid vector pET 28a-BcCR was successfully constructed and transfered into E.coli B121(DE3)plysS to overexpression the BcCR.The BcCR was successfully expressed E.coli B121(DE3)plysS/pET 28a-BcCR as soluble protein.The protein size was approximately 32 kDa.The enzymatic properties of the heterologous expression BcCR from B.cereus were investigated.The optimum reaction temperature was 57.5?,and the optimum reaction pH was 7.0.The enzyme was stable below 40?.No ordinary divalent metal ions can significantly promote the catalytic ability of the enzyme.The kinetic parameter of the BcCR to ethyl 4-chloroacetoacetate(COBE)are Km=1.85 mmol/L,and the maximum reaction rate Vmax=0.22 ?mol·min-1·mg-1.These enzymatic properties of the heterologous expression BcCR were basically consistent with the result of BcCR and ApCR alignment.The carbonyl compound Ethyl-4-chloroacetoacetate(COBE)was reduced to the corresponding chiral Ethyl-4-Chloro-3-Hydroxybutyrate(CHBE)catalyzed by the whole cells E.coli B121(DE3)plysS/pET 28a-BcCR.The reduced product was detected by gas chromatography.When the concentration of substrate was 20 mmol/L and the concentration of bacteria was 0.1 g/mL,the yield was 67.7%after reacted at 37? for 20 h.The optical rotation of the carbonyl reduction product is positive,which indicated that the product is(R)-CHBE.In this paper,a gene mining strategy and method for carbonyl reductase was developed.Using this method,a novel carbonyl reductase,BcCR,from B.cereus was successfully obtained,which provided basic research for obtaining high-efficiency carbonyl reductase.Compared with the traditional method of obtaining carbonyl reductase,gene mining appears to be more efficient and has a broader prospect.