| Epoxide hydrolase(EH,EC3.3.2.3)is a generic term for enzymes that catalyze the hydrolysis of epoxides to produce the corresponding vicinal diols,and belongs to the class of ?/? sheet hydrolases.Membrane-associated epoxide hydrolase(maEH)has played an important role in the formation of carcinogens and has received extensive attention as an early marker of liver cancer.Soluble epoxide hydrolase(sEH)regulates the hydrolysis metabolism of epoxy arachidonic acid and is involved in the regulation of blood pressure and inflammatory responses.Therefore,hypertension and atherosclerosis can be treated by designing some inhibitors similar to the structure of the substrate.Hardening,renal failure and other diseases.In addition,EHs with high enantioselectivity are a very valuable biocatalyst for hydrolytic kinetic resolution of chemically synthesized,inexpensive racemic epoxides to produce optics.Pure o-diols and epoxides are used to synthesize optically pure drugs with high efficacy and side effects.Therefore,EH is a biocatalyst with great industrial application value.The white-rot basidiomycete bacterium Phanerochaete chrysosporium preserved in our laboratory has been annotated with 20 epoxide hydrolase encoding genes have been annotated and 11 epoxide hydrolases have been cloned.Where PchEHA has the highest activity for the substrate epoxy styrene.In order to make full use of the encoded epoxide hydrolase gene resource,it was applied to the epoxide chiral resolution.In this study,use dITP as mutagen,endoV digest DNA random mutation of PchEHA gene from P.chrysosporium BKM-F1767 and construct a "gene mutation library" consisting of at least 100,000 independent clones.The mutation was evaluated and the results showed that the mutation rate of the mutation library constructed by this method was 100%,with an average of 5.09 mutation points per 1 kb,and an average of 5.13 base mutations per PchEHA gene.The pETEHA plasmid containing the mutant PchEHA gene was transformed into E.coliBL21 and induced using IPTG.Using(R)-Styrene oxide [(R)-SO] and(S)-Styrene oxide[(S)-SO] as substrates for high-throughput screening of 96-well plates.Primary screening was performed based on(R)-/(S)-SO hydrolase activity,>15,000 clones were screened,and more than 230 mutant strains with enhanced enzymatic activity or enantioselectivity were obtained.After 96-well rescreening,68 mutant strains were screened.These 68 strains were induced by IPTG in flasks and re-screened with(R)-/(S)-SO as substrate.42 strains of mutant strains were obtained.DNA sequencing was performed on these mutant strains and the strains were screened.There are amino acid changes in the PchEHA protein.Among them,21 strains preferentially hydrolyzed(R)-SO,6 strains preferentially hydrolyzed(S)-SO,and 14 strains increased the activity of(R)-/(S)-SO by 20% to 40%.Based on the mutation sites,mutation types,and number of amino acid mutations in the42 mutant strains,34 mutant strains were finally selected for protein expression,purification,and enzyme activity assay.Among the 34 mutants selected,3 mutants(M30,M33,M34)had a hydrolysis rate of 95% or more for(R)-SO,and the enzyme activity increased by more than60%;4 mutants(M8,M11,M12,M20)for(S)-SO hydrolysis rate of more than 40%,of which the M8 mutant degradation rate is the highest,is 47.74%,the enzyme activity increased by90%;The hydrolysis activity of(R)-SO in PchEHA wild type was 2.43 times that of(S)-SO.,but the mutant M7 has a hydrolyzed(R)-SO hydrolysis activity which is 7.97 times that of the(S)-SO and enhances the enantioselectivity.Mutant M30 had the highest hydrolyzed activity to glycidyl tosylate(GT)in both configurations,and its hydrolysis activity to(R)-/(S)-GT was78.87% and 24.00%,respectively,compared with wild-type.The activity of the enzyme increased by 1 and 3 times,respectively;4 mutants(M6,M14,M31,M32)showed markedly decreased(S)-GT activity and even showed inactivation,greatly improving the enantioselectivity.The rate of hydrolysis of(R)-epichlorohydrin [(R)-Ep] by the mutant M33 was 67.68%,and the enzyme activity increased by more than 30%;mutant M30 on(S)-epichlorohydrin [(S)-Ep],the hydrolysis activity was the lowest,the hydrolysis rate of(R)-Ep was 3.97 times higher than that of(S)-Ep,and the enantioselectivity increased more than 2 times.Ep as substrate,the enantioselectivity of the mutant M22 was shifted relative to the wild type,and the hydrolysis activity of(S)-Ep was superior to(R)-Ep.Through the analysis of 34 mutant enzyme activities and mutation sites,a total of 7mutants such as M2 and M9 were selected to carry out C108 S,Y163H,and E251 G site combination mutations,and 14 combination mutants were successfully constructed and expressed.By enzyme activity assay,the results showed that the combined mutants F2,F4,F6,and F8 all had a hydrolysis rate of(R)-SO above 90%;for(S)-SO,the combination mutantsF6 and F8 have more than a 1-fold increase in wild-type hydrolytic activity.14 strains of mutants had a hydrolysis rate of Rac-SO of 50% to 70%.The PchEHA wild-type preferentially hydrolyzed(R)-GT,the combined mutants F7 and F10 transformed the enantioselectivity of the hydrolysis GT,preferentially hydrolyzed(S)-GT.In order to further study the effect of combinatorial mutations on the PchEHA enzyme activity,kinetic curves were determined with SO as a substrate.The results showed that when(R)-SO was used as a substrate,the kcat/Km was increased in all 10 combinations(F1,F2,F4,F5,F6,F8,F9,F11,F13,F14).Among them,the kcat/Km values of the F4 and F6 combinatorial mutants were about 2 times that of the wild type.With the(S)-SO as substrate,the kcat/Km increased in the 6 combinations of mutants(F1,F2,F8,F11,F13,F14).The ratio of the catalytic efficiency(kcat/Km)of hydrolysis of(R)-/(S)-SO by epoxide hydrolase is called the E value,reflecting the enantioselectivity of the enzyme to the substrate in the catalytic reaction.Except that the E values of the four mutants(F1,F10,F13 and F14)were slightly lower than those of the WT,the E values of the other 10 mutants increased,indicating that the enantioselectivity of the 10 mutants was higher than that of the wild type.In this study,the PchEHA mutant library was successfully constructed using the dITP nucleotide analogue endoV endonuclease.The 96-well plate high-throughput screening and the triangular flask fermentation rescreening were used to obtain more than 40 mutants.Through the enzyme activity assay of 34 mutants,the results showed that when styrene oxide and other epoxides were used as substrates,the enzyme activity and enantioselectivity of the mutants were significantly increased.The combination of the mutation sites also further improved the enzymatic activity and enantioselectivity of PchEHA,which provided a sufficient experimental theoretical basis for the industrial application of PchEHA. |
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