Expression And Engineering Of Halohydrin Dehalogenase And Its Application In The Synthesis Of Ethyl(R)-4-cyano-3-hydroxybutyrate

Halohydrin dehalogenase（HHDH）catalyzes the dehalogenation of vicinal halohydrins to form epoxides,accompanying the release of halide anions and protons.On the other hand,epoxides could be converted to corresponding?-substituted alcohols by HHDH in the presence of nucleophiles.HHDH played an important role in the preparation of optically active halohydrins,expoxides and?-substituted alcohols because of its high efficiency,enantio-selectivity and region-selectivity.Ethyl（R）-4-cyano-3-hydroxybutanoate（HN）is a key chiral building block for a salable cholesterol-lowering drug of atorvastatin（Lipitor）.The HHDH-mediated reaction for preparation of HN occured in mild conditions with brief processes and gave high yield,which received more and more researchers‘attentions.In this thesis,the research work was carried out focusing on the process for HN by using HHDH as the biocatalyst:Six short-chain dehydrogenase/reductase（SDR）genes were mined,cloned and expressed for HHDH activity analysis.The SDR6 gene from Parvibaculum lavamentivorans DS-1 expressed in E.coli BL21（DE3）/p ET-32a（+）system showed HHDH activity.Alignment analysis showed that SDR6 shared approximately 30%sequence similarity with the other reported HHDHs,which indicated SDR6 was a novel HHDH（named Hhe D）.In addition,Hhe D was purified for the studies of substrate specificity,effect of metal ions on the activity,and the kinetic resolution of epoxides.Moreover,three characterized HHDH(Hhe A_AD2,Hhe B_GP1 and Hhe C)genes were synthesized and expressed to construct an enzyme library with Hhe D.The process for（S）-4-chloro-3-hydroxy-butyronitrile[（S）-CHBN]from 1,3-dichloro-2-propanol（1,3-DCP）by HHDH was investigated.After screening the enzyme library,Hhe C was identified with high activity and enantio-selectivity in the synthesis of（S）-CHBN.Through reaction optimization and site-directed mutagenesis,mutant W249F was obtained and applied to conversion of 10 g/L 1,3-DCP,which generated97.5%ee and 86%yield of（S）-CHBN.In addition,biotransformation of20 g/L 1,3-DCP was achieved by co-catalysis of mutant W249F and W249F/W139D,which gave the（S）-CHBN with 96.9%ee and 86%yield.The process to prepare HN from（S）-4-chloro-3-hydroxybutanoate[（S）-CHBE)]by HHDH were also studied and Hhe D were found with highest efficiency among the enzyme library.After optimization of the reaction conditions,8.0 and 40 ^oC were identified were the optimal p H and temperature for preparation of HN.On the optimal condition,200 g/L（S）-CHBE were converted to HN by whole-cell catalysis of 40 g DCW/L Hhe D,which generated 95%conversion and 85%yield in 14 h.A novel high-throughput method for detection of HHDH activity was developed based on the determination of the azides and applied to directed evolution of Hhe D.A random mutant library was constructed by error-prone PCR.After screening 2,500 colonies,three improved mutants F176M,A187R and A187S were identified with 2.1,1.8 and 1.6-fold increased relative activity,respectively.By combining the substitutions,two variants of F176M/A187R and F176M/A187S were obtained and respectively showed 3.1 and 2.3-fold of activity than the wild type of Hhe D（Hhe D-WT）.Subsequently,Hhe D-WT and F176M/A187R were expressed and purified to determine the kinetic parameters.The results indicated the―best‖variant F176M/A187R displayed 2.8 times of efficiency than Hhe D-WT in the synthesis of HN from（S）-CHBE.