| Halohydrin dehalogenase(HHDH)is a multifunctional biocatalyst that can be used to synthesize chiral epoxides and β-substituted alcohols.In addition,HHDHs play a key role in biodegradation of halogenated organic compounds in terms of environmental management.Although more and more HHDHs have been discovered and identified,natural HHDHs thus far rarely exhibit high activities and high stereoselectivities toward a few selected substrates,which greatly limits their practical applications.Hence,the development of HHDHs with excellent catalytic performance and application potential is always considered to be a focused issue in the HHDHs-mediated biocatalyis research.In this study,we focused on the research of a novel halohydrin dehalogenase(HHDH-Ra)from Rhodospirillaceae bacterium.Directed evolution and semi-rational design strategy were adopted to obtain the active HHDH-Ra mutants with enhanced catalytic activity and enantioselectivity toward the model substrates,such as 1,3-dichloro-2-propanol(1,3-DCP)and(R/S)-2-chlorophenylethanol(2-CPE).The main conclusions are as follows:1.The mutant HHDH-Ra-A138S with improved activity obtained in our previous study was chosen as the starting template.A random mutant library was constructed by error-prone PCR,and 13 mutants which showed enhanced enzyme activities toward 1,3-DCP were identified.The enzyme activities of these mutants were 1.08-1.89 folds of HHDH-Ra-A138S.Among them,four mutants(A194T/K201E,R124C,T21A,and R73C/M199V)with higher activities were selected for subsequent combinatorial mutagenesis,and the combinatorial mutants R73C/M199V/T21A and R73C/M199V/R124C with further improved activity were obtained.The enzyme activities of R73C/M199V/T21A and R73C/M199V/R124C were 2.5 and 2.75 folds of HHDH-Ra-A138S,respectively.2.The crystal structure of HHDH-Ra with 1.97(?) high-resolution was resolved through X-ray diffraction.Based on the molecular docking of the mutant,A138S/R73C/M199V/R124C(named as Mu-S4)and 1,3-DCP,the polar/nonpolar scanning and site-sautrated mutagenesis were performed on Mu-S4,and two key sites of A88 and N180 which affected catalytic activities and stereoselectively were obtained.Then,the site-sautrated mutagenesis was performed on wild-type HHDH-Ra,five mutants(A88K,A88R,A88M,N180G and N180A)with inverted stereoselectively were obtained.Meanwhile,eight mutants(A88N,N180M,N180L,N180C,N180H,N180F,N180Y and N180I)with improved stereoselectivity were also obtained,among them,the ee value of N180M catalyzed product(S)-ECH increased from 13.11%of wild type to 90.54%.Structural analysis of the mutant N180M revealed that the increase stereoselectively was mainly attributed to the fact that inner C atom of the substrate molecule formed a larger steric hindrance and a new hydrogen bond,which resulted in the stronger binding of the chlorine atom connected to the inner C atom.3.Alanine scanning and site-sautrated mutagenesis were employed to engineer the activity and stereoselectivity of HHDH-Ra toward substrate(R/S)-2-CPE.Two mutants N180A and N180G with significantly improved activities toward S-2-CPE were obtained,whose activities were 9.5 and 15 folds of wild-type HHDH-Ra,respectively.Additionally,the ee value of(S)-SO produced from S-2-CPE by mutant N180G was increased to≥99.9%compared with the wide-type HHDH-Ra of 89.11%.Then,the mutants N180A and N180G were used for the kinetic resolution of(R,S)-2-CPE,resulting in the eep value of the product(S)-SO reached 91%and 97.17%,respectively,and the enantioselectivity(E value)of mutant N180A and N180G were increased from 1.51 of wile-type to 71.75 and 278.Analysis of variant(N180A,N180G)structures demonstrated that increased activity was due to the enhanced binding force between the enzyme with the substrate and the deprotonation process of Tyr149 acting on the substrate hydroxyl group was more easier occur.Besides,the increased stereoselectivity was attributed to the steric hindrance effect,and then caused the change in the angle of the benzene ring located at the catalytic pocket. |
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