A Study On Enzymatic Properties And Evolution Of Halogen Aldol Dehalogenase

Halogen alcohol dehalogenase catalyzes the dehalogenation of?-halo alcohols to form chiral epoxides by an intra-molecular nucleophilic substitution mechanism,and can catalyze the ring opening of epoxides to form chiral?-substituted alcohols under different unnatural nucleophile-mediated conditions.Halo alcohol dehalogenases can be used in the synthesis of high-value chiral pharmaceutical intermediates such as chiral epoxides and chiral?-substituted alcohols.In addition,the haloalcohol dehalogenase can also degrade heterogeneous halides.Certainly,halogen alcohol dehalogenase has a high application value in environmental management.Therefore,in this research work,the high-efficiency expression,enzymatic properties,application and molecular modification of a newly discovered halohalogen dehalogenase HHDH_XF2F2 were mainly studied.The specific results are as follows:（1）The experimental results show that the optimal expression condition of the halo-alcohol dehalogenase HHDH_XF2:when the OD₆₀₀=1.0,the final concentration of0.05 mmol/L IPTG is added and induced at 24℃,150 rpm for 10 h,the halo alcohol is dehalogenated.At this time,the enzyme activity expressed by the halogen alcohol dehalogenase can reach up to1438.25 U/mL.（2）Resuiting enzymatic properties of halohydrin dehalogenase HHDH_XF2 with1,3-dichloropropanol as substrate.The optimum reaction temperature was 47°C and the optimum pH was 8.67.It was stable in within temperature range of 35-40°C,and with good promoting effect on the catalytic reaction when the concentration of Zn²⁺was 0.8mmol/L.The kinetic parameter of the enzyme for 1,3-dichloropropanol was K_m=0.035mmol/L,and the maximum reaction rate V_max=0.0025 mol/min.（3）The halogenated alcohol dehalogenase HHHDH_XF2 was constructed by using error-prone PCR aimed to construct a mutant library,and the library was screened by colorimetric method.Finally,a positive mutant with a 6.6-fold increase in stereoselectivity was screened.（4）The study show that halohydrin dehalogenase HHDH_XF2 catalyzed the formation of epichlorohydrin by 1,3-dichloropropanol,the optimum catalytic conditions were found to be:optimum pH=8.34,optimum temperature of 45℃and substrate concentration of30 mmol/L.After 2 h of conversion under these conditions,the highest conversion of1,3-dichloropropanol was 89.4%.It was found that the halo-alcohol dehalogenase HHDH_XF2F2 degraded beta-cypermethrin.When the degradation temperature was 45℃,the degradation rate of beta-cypermethrin could reach 41.2%after 2 h degradation.