Molecular Modification And Functional Characterization Of Ethyl Carbamate Hydrolase

Ethyl carbamate(ethyl carbamate,EC)is a trace toxic and harmful substance produced during the production,transportation and storage of fermented food,which has potential carcinogenicity and genetic toxicity to human body.The EC properties are relatively stable,and it is difficult to remove by physical and chemical methods.Using EC hydrolase to hydrolyze EC into ethanol is a reliable and effective method.However,EC hydrolase has been difficult to be applied in industry due to the problems of poor ethanol tolerance and stability.Therefore,in this study,an EC hydrolase from Bacillus lysine was modified to solve the above problems.The specific research contents are as follows:Firstly,EC hydrolase was modified by diphasic high pressure molecular dynamic simulation(d HP-MD),and positive single mutant proteins were obtained.The loop regions with large differences in isothermal compression coefficients were selected as the sensitive region which were used to transformed.The free energy changes of the mutants in the sensitive regions were calculated by Fold X and I-mutant,and the potential mutants with improved stability were finally obtained as:A321I,S325F,S325Y,S325N,Q332E,Q332G,H68A,H68L,H68M,H68K,H68Y,K70R and A178L.Experimental verification showed that H68A,K70R,A178L and S325N had better enzymatic properties,and their specific enzyme activities were 2.78 times,1.78 times,2.84 times and 2.21 times of that of WT.At 10%(v/v)ethanol concentration,the relative enzyme activity of H68A,S325N,K70R and A178L was 1.31 times,2.63 times,1.81times and 1.78 times higher than that of WT,respectively.Secondly,it was verified that combination mutation could improve the enzymatic properties of EC hydrolase,and the reasons for the improved ethanol tolerance was analyzed.It was found that the specific enzyme activity of triple mutant H68A/K70R/S325N with the best ethanol tolerance was about 3.41 times that of WT,and its relative enzyme activity was 5.02times that of WT under 20%(v/v)ethanol concentration,and its T_m value increased to 54.9°C.In order to analyze the reasons of the improvement of ethanol tolerance,H68A/K70R/S325N was simulated in an environment with 1 bar and 50%(v/v)ethanol concentration.The results showed that near the mutation sites a new hydrogen bond was formed and the fluctuation has also decreased there,the overall structure of the protein was tightened to resist the interference of ethanol,and a more complete hydration layer was formed outside of the protein.In addition,the number of ethanol molecules in 70R,325N and near the active center decreased,indicating that the ethanol tolerance of the mutant was improved.Finally,EC hydrolase was immobilized to further improve its enzymatic properties and the immobilization conditions was also optimized.It was found that the immobilization condition was let the chitosan pellets activated by 1%(v/v)glutaraldehyde for 3h and crosslinked with the enzyme solution for 4h.After immobilization,the ethanol tolerance of H68A/K70R/S325N was 1.85 times that of the free enzyme,and the enzyme activity of H68A/K70R/S325N was also increased to 28.30%at 55°C.In the fifth reaction of the reuse experiment of EC hydrolase,the immobilized pellets still had 54.31%relative enzyme activity.The immobilized and free H68A/K70R/S325N degraded 28.62%and 14.81%EC in simulated wine samples,respectively.In conclusion,the mutant H68A/K70R/S325N with improved enzyme activity and ethanol tolerance was effectively obtained by d HP-MD method,and its potential application on an industrial scale was further explored through immobilization,which could lay a foundation for the subsequent modification of EC hydrolase.