Improving The Regioselectivity Of RpEH For M-Nitrostyrene Oxide Based On Semi-Rational Design

Epoxide hydrolases can catalyze the asymmetric hydrolysis（regio-or enantioselectivity hydrolysis）of racemic（Racemic,rac-）epoxides to produce optically 1,2-diols or epoxides.Optically epoxides and 1,2-diols are highly value-added synthons in the field of biomedicine with extremely high economic benefits,promoting the development of catalytic research on EHs.Currently,most wild-type EHs have poor stereoselectivity,resulting in low yields or optical purity of the products.Therefore,improving the stereoselectivity of the EHs with poor catalytic performance based on semi-rational design,is an important direction of current EHs research.A novel epoxide hydrolase RpEH gene was cloned from Rhodotorula paludigena,and successfully expressed in Escherichia coli BL21（DE3）in our laboratory.On the basis of previous research,the main research contents and results are as follows:（1）The whole-cell specific activity and regioselectivity of RpEH towards rac-1a were determined.The whole-cell specific activity of RpEH towards rac-1a was 85.4 U·g^-1 wet cell,the regioselectivity coefficientsα_S andβ_R were 92.2%and 95.2%,respectively,and the ee_p value of（R）-1b was 87.5%,which means that RpEH has the potential for enantioconvergent hydrolysis towards rac-1a.Therefore,rac-1a was selected as the model substrate to engineering the regioselectivity of RpEH based on computer-aided design.（2）The crystal structure of Aspergillus niger EH（An EH,PDB:1QO7）with 39.4%primary structural sequence homology to RpEH was selected as the template for homology modeling of RpEH.Based on the molecular docking results of RpEH and（S）-1a,8 residues in the substrate binding pocket（SBP）of RpEH that have intermolecular forces with（S）-1a were selected for valine scanning mutagenesis.The whole-cell specific activity of the 8 valine scanning mutants towards rac-1a and the ee_p value of（R）-1b were determined.After preliminary screening,it was found that the whole-cell specific activities of RpEH^L360Vand RpEH^F361Vtowards rac-1a were increased significantly(156 and 117.6 U·g^-1 wet cell),about 1.8 and 1.4 times than that of RpEH,while the whole-cell specific activity of the other 6 mutants were decreased(2.0～70.8U·g^-1 wet cell).RpEH^Y254V and RpEH^L360V catalyzed the hydrolysis of rac-1a to produce（R）-1b with improved ee_p values from 87.5%to 92.0 and 94.8%,respectively.The ee_p values of（R）-1b catalyzed by the other six mutants were decreased（49.6～87.3%）.In view of the above experimental results,the ee_p value of（R）-1b was used as the screening criterion,RpEH^Y254Vand RpEH^L360V with improved ee_p values were selected for further study.（3）Tyr²⁵⁴ and Leu³⁶⁰ were selected for saturation mutations,respectively.Among the 19mutants at Tyr²⁵⁴,RpEH^Y254D and RpEH^Y254I showed no activity,while the ee_p value of（R）-1b of the other 17 mutants increased（88.4～94.3%）,and RpEH^Y254P increased significantly from87.5%（RpEH）to 94.3%.Among the 19 mutants at Leu³⁶⁰,the ee_p values of（R）-1b were all improved significantly（88.8～95%）,and the ee_p values of（R）-1b that catalyzed by RpEH^L360Cwas highest,which was 7.5%higher than that of RpEH,the whole-cell specific activity of RpEH^L360C for rac-1a was 43 U·g^-1 wet cell,and the regioselectivity coefficientsα_S andβ_R were determined to be 96.3%and 98.6%,respectively.In order to obtain mutants with higher regioselectivity,RpEH^Y254P and RpEH^L360C were selected for iterative saturation mutations.Among the 19 RpEH^L360C/Y254X mutants,all ee_p values of（R）-1b catalyzed by RpEH^L360C/Y254Xwere improved（94.2～96.8%）except RpEH^L360C/Y254F and RpEH^L360C/Y254P,which completely lost their activity.Among the 19 RpEH^Y254P/L360X mutants,the ee_p values of（R）-1b catalyzed by RpEH^Y254P/L360V was highest（96.9%）and the whole-cell specific activity of RpEH^Y254P/L360V for rac-1a was determined to be 3.7 U·g^-1 wet cell,while the other 18 mutants showed lower activity or regioselectivity,the ee_p values of（R）-1b that catalyzed by the 18 mutants were32～96.5%.Therefore,considering the activity and the ee_p values of（R）-1b,the best mutant was selected to be RpEH^L360C.（4）The maximum allowable concentration of rac-1a that catalyzed by RpEH^L360C in phosphate buffer was 100 mmol·L^-1.After screening the addition of Tween-20,it was found that the maximum allowable concentration of rac-1a increased to 600 mmol·L^-1 in 30%Tween-20/phosphate buffer.The scale-up preparation of（R）-1b with 93.4%ee_p,95.7%yield and 42.1g·L^-1·h^-1 was performed in this condition.（5）The catalytic properties of RpEH^L360C for other epoxides with different substituent groups were determined.Among them,RpEH^L360C performed high activity(129～610 U·g^-1 wet cell)toward rac-2a,5a,6a,8a and 9a,and high regioselectivity toward rac-6a and 9a,producing products with ee_p values of 92.4 and 92.0%,it also showed high enantioselectivity for rac-4a with an E value of 40.