| Carbonyl reductase belongs to the first subclass of oxidoreductase,which is widely found in bacteria,fungi,yeast,plants and animals.It is a kind of cytoplasmic enzyme with broad substrate specificity.Carbonyl reductase can catalyze the asymmetric reduction of a series of carbonyl compounds such as aliphatic ketones,aromatic ketones,and aldehydes in the presence of coenzyme NAD（P）H to obtain the corresponding chiral alcohols.Chiral alcohols containing chiral hydroxyl functional groups are important chiral building blocks in organic synthesis and are widely used in pharmaceutical intermediates,fine chemicals and other fields.The biocatalytic method for asymmetric reduction of carbonyl reductase to chiral alcohols has great potential for industrial application.In our previous study,the recombinant strain E.coli BL21（DE3）/p ACYC Duet-1-cpcr was constructed.On this basis,the carbonyl reductase Cp CR was molecularly modified to expand its substrate spectrum and enhance its catalytic efficiency for the target substrate.A random mutation library was constructed by error-prone PCR,and 2,4-dinitrophenylhydrazine（DNPH）high-throughput screening enzyme catalyzed phenylacetaldehyde（PAAH）,（4-chlorophenyl）（2-pyridyl）methanone（CPMK）and other ketone substrate mutants.Molecular modification of enzymes was studied by computer-aided design.Site-directed mutagenesis to construct mutant enzyme.The enzymatic properties of the mutants were explored,including the catalytic activity,thermal stability,kinetic parameters,etc.The effects of catalytic time,temperature,p H,substrate concentration,glucose concentration and organic cosolvent on the enzymatic conversion of PAAH/CPMK were investigated.The main experimental results are as follows:（1）A high-throughput screening method for carbonyl reductase Cp CR activity was established,that is,the characteristic reaction of the carbonyl chromogenic agent DNPH and the substrate containing the carbonyl group to generate red-brown precipitate was used for high-throughput screening,and it was applied to the screening of mutant libraries during the molecular modification of reductase Cp CR.（2）Molecular modification of PAAH catalyzed by carbonyl reductase Cp CR:Molecular docking of carbonyl reductase and PAAH was performed by Discovery Studio software.The key amino acid sites of enzyme binding to substrate were analyzed.Combined with the positive mutation sites screened by error-prone PCR,amino acid substitution,virtual saturation mutation and mutation energy calculation were performed.The amino acids with the lowest mutation energy were selected for site-directed mutagenesis to construct S50N,Q60W,A97K,L165D,T171F,P284E and L309W mutants.The enzymatic properties of the mutants T171F and L165D showed that the enzyme activities were 11.46 U/mg and 11.96U/mg,respectively,which were 10.14%and 12.86%higher than that of wt Cp CR.Kinetic parameter experiments showed that the carbonyl reductase wt Cp CR and its mutants had high affinity for PAAH,and the catalytic efficiency of T171F was twice that of wt Cp CR.In the thermal stability experiment of the enzyme,the t1/2of T171F was increased to 28 h compared with wt Cp CR by 7 h.The T50value of T171F is 37°C,which is 1°C higher than that of wt Cp CR.The yield of T171F was 91.31%,which was 2.36 times higher than that of wt Cp CR.It indicated that the mutant T171F had stronger catalytic ability and thermal stability.The factors affecting the synthesis of 2-PE catalyzed by mutant T171F and wt Cp CR were investigated.The results showed that the optimum catalytic temperature was 30°C.The optimum p H of T171F was 6.5,and the optimum p H of wt Cp CR was 7.0.When the concentration of PAAH was 1000 mg/L,the yield of T171F was more than 90%,while wt Cp CR was only 31.7%.The optimum glucose addition was 2.0 g/L;when the substrate concentration was 2000 mg/L,the yield of T171F in 5%（V/V）dimethyl sulfoxide solvent was the highest,up to 85.08%,which was 3.1 times that without organic cosolvent.Compared with wt Cp CR,the catalytic time of T171F was reduced from 10 h to 4 h,and the catalytic efficiency was greatly improved..（3）Molecular modification of CPMK catalyzed by carbonyl reductase Cp CR:The mutant sites obtained by high-throughput screening were combined with computer-aided design to construct mutant enzymes A97N,A121N,K289D,P284E/P285D and P284D/P285W.The enzymatic properties showed that the enzyme activity of the mutant P284D/P285W was 0.6266 U/mg,which was 3.42 times that of wt Cp CR.Kinetic parameter experiments showed that the affinity of carbonyl reductase wt Cp CR and its mutants to CPMK was low,and the catalytic efficiency was 6.2 times that of wt Cp CR.In the thermal stability experiment of the enzyme,the t1/2of A97N was increased to 24 h compared with wt Cp CR by2 h.The T50value of P284D/P285W is 38°C,which is 2°C higher than that of wt Cp CR.The experiment of catalyzing CPMK to synthesize（S）-（4-chlorophenyl）（2-pyridyl）methanol[（S）-CPMA]showed that the conversion rate of P284D/P285W was 22.34%,which was 4.35times that of wt Cp CR.However,its e.e.value decreased to 81.09%.The mutant P284D/P285W has stronger catalytic ability and thermal stability.The influencing factors of CPMK catalyzed by mutant P284D/P285W and wt Cp CR were investigated.The results showed that the optimum catalytic temperature of wt Cp CR was32°C,and P284D/P285W was 36°C.When the temperature exceeds 36°C,the stereoselectivity of the enzyme decreases and the e.e.value decreases.The optimum catalytic p H of the enzyme was 7.5,and the change of p H had no effect on e.e.value.When the concentration of CPMK was 100 mg/L,the conversion rate of P284D/P285W was 42.71%,while wt Cp CR was only 10.25%.The optimum glucose addition was 3.0 g/L.Compared with wt Cp CR,the catalytic time of mutant P284D/P285W was reduced from 8 h to 6 h,and the catalytic efficiency was improved. |
