The Researches In Heterologous Expression,Molecular Modification And Application Of Solanum Lycopersicum Epoxide Hydrolases

Epoxide hydrolases(EHs),can catalyze the kinetic resolution or enantioconvergent hydrolysis of epoxides,producing enantiopure epoxides or diols.The chiral epoxides and diols,highly valued-added synthons,are used for the syntheses of a varity of compounds with complicated bioactive functions and playing critical roles in pharmaceutical industries with considerable economic benefits.Correspondingly,the processes of EHs'catalysis research are accelerated for the applications of these versatile building blocks.However,there have some problems in the EHs'studies,such as narrow substrate scopes and unsatisfactory stereoselectivities of an EHs.To solve these problems and provide more ideal options for the syntheses of enantiopure epoxides and diols,in this work,a series of researches have been studied.Firstly,based on the bioinformatics analysis,two EHs were coloned from Solanum lycopersicum and heterologously expressed in E.coli BL21.Secondly,the enzymatic properties and substrate spectrograms of the two EHs were assayed,respectively.Finally,according to the application bottlenecks of each EHs,further studies were separately carried out.The results were as follows:(1)Based on the bioinformatics analysis,two genes,sleh1 and sleh2,were coloned from Solanum lycopersicum,which were encoding 321 amino acids of Sl EH1 and Sl EH2.Sl EH1 and Sl EH2,belonging to?/?-hydrolase family,were two novel EHs from plant.The heterologous expression of two enzymes were performed by constructing E.coli transformants,E.coli/sleh1and/sleh2,followed by confirming their appropriate incuced temperature,time and IPTG concentration of 25?,8 h and 0.05 m M,respectively.The two enzymes were purified and the p H optimum,stability and temperature stability of Sl EH1 were separately determined to be7.0?7.5,6.5?8.5 and 40?.(2)The application potentials in syntheses of chiral epoxides and diols of Sl EH1 and Sl EH2were analyzed,from activities,antioselectivities and regioselectivities,et al.Sl EH1 displayed the extremely high E value(>200)towards rac-2a which was obviously higher than those of EHs previously reported and potentials for enantioconvergent hydrolysis of rac-11a,generating(R)-11b with ee_p of 51.3%.Sl EH2 exhibited the property of enantioconvergent hydrolysis of rac-1a,which catalyzing the hydrolysis of 20 m M rac-1a affording(R)-1b with 87.1%ee_p and94.4%yield.(3)To enhanced the?_S of Sl EH1 for rac-11a,the modification of Sl EH1 was performed.Ten specific residues lining the substrate-binding pocket of Sl EH1 were identified based on the molecular docking simulation of Sl EH1 with rac-11a.After constructing and screening of several mutants,one double-site mutant,Sl EH1^W106T/F189L,was obtained with increased?_S from55.3%from 96.7%,affording(R)-11b with enhanced ee_p from 51.3%to 95.4%.The gram-scale enantioconvergent hydrolysis of 400 m M rac-11a was carried out using E.coli/sleh1^W106T/F189Lwet cells at 20°C for 24 h,producing(R)-11b with 94.7%ee_p and 95.6%yield.The substrate spectrum assay of Sl EH1^W106T/F189L shown that it producing(R)-12b with increased ee_p from9.5%to 83.0%,while(R)-14b with high ee_p of 97.4%.(4)To enhance the substrate loading of enantioconvergent hydrolysis of rac-1a catalyzed by E.coli/sleh2,the reaction medium was optimized.The results indicated that the substrate loading for the effective hydrolysis was enhanced from 200 m M to 400 m M,in the 30%(v_s/v_b)tween-20/phosphate buffer system catalyzed by E.coli/sleh2.The gram-scale preparation of(R)-1b with 96.2%ee_p and 97.2%yield was performed,in the best reaction system.The 30%(v_s/v_b)tween-20/phosphate buffer medium was determined to have wide scopes,in which the c of enantioconvergent hydrolysis of 350 m M rac-5a catalyzed by E.coli/pveh1^Z6 was increased from 44.9%to>99%,and 500 m M rac-m NSO catalyzed by E.coli/rpeh^L360C was increased from 67.7%to>99%.