Improving The Enzymatic Activity Of Halohydrin Dehalogenase(Hhec) Toward 2,3-dcp Using Combined Directed Evolution Strategies

1,2,3-Trichloropropane(TCP) is an industrial chemical waste that has been formed in large amounts during epichlorohydrin manufacture.It is a persistent groundwater pollutant and a suspected human carcinogen.There is a need for cheap and efficient technologies for the cleanup of TCP-contaminated sites.Compared with the conventional degradation techniques,biodegradable pollutants approach is more environmentally-friendly and efficiently,it also can reduce cost and bring further benefits.Halohydrin dehalogenases are lyases that catalyze the cleavage of carbon-halogen bond of halohydrins.They also can catalyze the reverse reaction in the presence of nucleophiles.HHDHs have been recognized as the ideal tools for the degradation of various halogenated environmental pollutants.Moreover,they can be used as biocatalysts for the kinetic resolution of halohydrins and epoxides,and for the preparation of various substituted alcohols.The stereoselectivity and catalytic activity of halohydrin dehalogenase are two important aspects of this paper.Recent studies show that the degradation of an intermediate2,3-dichloro-1-propanol(2,3-DCP)catalyzed by HheC greatly limited the biodegradation rate of TCP in a multi-enzyme system,thus,a more active HheC mutant related to wild-type HheC is desired.In this paper,we used the combined transformation method to improve the catalytic activity of HheC enzyme toward2,3-DCP.Firstly,the information of key amino acid residues related to dehalogenation of 2,3-DCP was obtained by computer method.We predicted 10 key sites that may play a role in enzymatic activity.By using a semi-rational design strategy,6 structure-guided libraries have been constructed and about 8200 colonies have been screened towards activity and enantioselectivity.30 positive mutants have been identified.Among them,,the best mutant P84 A showed an 21.8 fold improved in activity.,F86 I,F12Q/F186 L,and W249 P exhibited a 16.67 fold,17.4 fold and 16.9 fold increased in activity,respectively.As for enantioselectivity,F86S/L142 Y showed a highly enantioselectivity(ER = 65 to ER = 200)and another one(W139A/Y187)with improved enantioselectivity(ER =65 to ER = 184).P84 A also showed an improved enzntioselectivity from ER = 7.6to ER = 72.Further analysis showed that amino acids P84,F86,F12,F186 and W249 werevery important to improve the activity of HheC toward 2,3-DCP.A positive effect on the enzyme activity was obtained when these residues were mutated to smaller amino acids.Amino acids P84,F86,L142,T134,N176 and W139 played a key role in regulating the stereoselectivity of the HheC toward the substrate.When P84,F86 and W139 mutated into small side chain amino acids,the halohydrin dehalogenase HheC showed a high preference with(R)-2,3-DCP,while such a preference was lost when T134 and N176 were mutated.In summary,the combined transformation strategy has a great advantage on modulating HheC with desired catalytic properties.