Molecular Modification Of Alcohol Dehydrogenase From Lactobacillus Kefir To Improve Its Catalytic Activity Towards Asymmetric Synthesis Of (S)-CHOH

(S)-tert-butyl-6-chloro-5-hydroxyl-3-oxohexanoate((S)-CHOH)is the key chiral intermediate of statins.Asymmetric reduction of tert-butyl-6-chloro-3,5-dioxohexanoate(CDOH)to(S)-CHOH catalyzed by alcohol dehydrogenases is a promising method.Nevertheless,the main problems is the low catalytic activity towards CDOH.MF147-A202L,a,mutant of alcohol dehydrogenase LkADH form Lactobacillus kefir DSM 20587 with the highest activity towards CDOH was researched in this study.The goal of this research was to improve the enzyme activity by molecular modification.The main contents of this work were as follows:1)First applying saturation mutagenesis at key sites(147,202)of MF147L-A202L,MF1471-A202L was obtained with improved specific activity.The specific activity reached 23 U/mg,which demonstrated 0.278-fold improvement in specific activity over MF147L-A202L.The mutants with improved enzyme activity were selected to do the combined mutantion.No mutants showed higher enzyme activity than MF147I-A202L.2)In order to improve the catalytic activity of MF147I-A202L,we constructed MF147I-A202L-CDOH complex by homologous modeling and molecular docking,and according to LkADH's structure and catalytic mechanism,we chose amino acid residues in the substrate pocket(G145,L153,Y190,1144 and G189)to do saturation mutagenesis,and MF147I-A202L-Y190F was obtained with improved specific activity.The specific activity reached 33 U/mg,which demonstrated 0.43-fold improvement in specific activity over MF147I-A202L.And then,virtual alanine scanning was applied at amino acid residues in the substrate pocket,and 5 mutants(M141A,S96A,L195A,V196A and M206A)was selected according to the mutation energy.MF147I-A202L-S96A was obtained with improved specific activity.The specific activity reached 28 U/mg,which demonstrated 0.217-fold improvement in specific activity over MF147I-A202L-Comibined MF147I-A202L-Y190F and MF147I-A202L-S96A we obtained MF147I-A202L-Y190F-S96A,The specific activity reached 44 U/mg,which demonstrated 0.91-fold and 1,44-fold improvement in specific activity over MF147I-A202L and MF147LA202L.3)A high throughput screening method based on fluorescence intensity of coenzyme was established,and by means of Error-Prone of alcohol dehydrogenase MF147I-A202L,a mutant was obtained with improved specific activity.Sequencing results showed that the site 190 of mutant was transformed from tyrosine to phenylalanine,and this mutant was also found in semi-rational design,which showed that the directed evolution method was effective,but a lot of screening work is needed before we can get a mutant with higher enzyme activity.