Rational Design Of BioH And Optimization Of Asymmetric Hydrolysis Of 3-cyclohexene-1-carboxylate

With the rapid increase in the market demand for chiral drugs,the preparation of chiral drugs and their intermediates has attracted more and more attention.S-3-cyclohexene-1-carboxylic acid is an important chiral precursor for the anticoagulant edoxaban,so the preparation of optically pure S-3-cyclohexene-1-carboxylic acid is of great significance.At present,its industrial production route is chiral resolution of 3-cyclohexene-1-carboxylic acid with chemical methods.Considering the disadvantages of chemical methods such as cumbersome procedures and the use of toxic reagents,exploration of simple and efficient biocatalytic methods that can replace chemical methods is important for the sustainable production of the anticoagulant edoxaban.The esterase BioH can catalysis the hydrolysis of ester bond and can be used for asymmetric hydrolysis of racemic methyl S-3-cyclohexene-1-carboxylate.However,the wild-type BioH showed low selectivity toward methyl S-3-cyclohexene-1-carboxylate(ee_p=32.32%),resulting in unsatisfactory yield and optical purity of the target product.Therefore,this thesis engineered the esterase BioH for improved enantioselectivity so as to develop a biocatalyst for preparation of S-3-cyclohexene-1-carboxylic acid.In this paper,using computational simulation as an auxiliary tool,rational design of esterase BioH was carried out by means of molecular docking and enzyme-substrate docking conformation,and strategies for selectivity improvement were developed.The steric hindrance of active pockets,the aromatic interaction and the hydrogen bonding interaction were adjusted to improve the S-selectivity.After obtaining the final mutant,molecular dynamics simulation was carried out for the final mutant and the wild-type esterase BioH,and the mechanism of selectivity improvement was discussed.The research contents and experimental results are detailed as follows:(1)Based on the understanding of the molecular structure and catalytic mechanism of the esterase BioH,BioH was docked with the substrate,and rational design was conducted based on the difference in the conformation of the enzyme-substrate complexes.6 out of the 9 single-point mutants constructed were positive mutants with improved selectivity for methyl S-3-cyclohexene-1-carboxylate,and the optimal mutant Mu3(L24A/W81A/L209A)had an ee_p value of 70.92%.(2)Soluble expression analysis of the above constructed mutants revealed inclusion bodies in all cases.In particular,Mu3 had only a small amount of soluble expression before optimization of the induction conditions,and could not reach the enzyme concentration required for subsequent reactions.Therefore,the expression conditions were optimized,and IPTG concentration of 0.1 mM,16°C,and TB medium induction for 24 h were found as the optimal induction conditions.(3)In order to improve the catalytic efficiency of the optimal mutant Mu3,the effects of temperature,pH,type and concentration of cosolvent on the catalytic reaction were investigated.The optimum temperature was 30°C,the optimum pH was 8.0,and the optimum solvent was 2.5%(v/v)Tween 80.The catalytic activity of Mu3 after optimization was about 2 folds higher than that before optimization.(4)Molecular dynamics simulation analysis was conducted for the wild-type esterase BioH and the positive mutant Mu3,and the reasons for the increase of S-selectivity for Mu3 were analyzed from the perspective of binding energy and conformation.It was found that the difference in binding energy between Mu3-R-substrate and S-substrate was higher than that of WT,leading to an increase in Mu3 selectivity.However,the binding energies of Mu3 to R-substrate and S-substrate were also higher than those of the WT,and the excessive active pocket after mutation was also unfavorable for substrate binding,resulting in decreased Mu3 enzyme activity.