| Dichloromethane(DCM)is a highly volatile compound and widely used as an industrial solvent.It is stable with a half-life over 700 years in aqueous solution and about 150 days in the atmosphere.DCM causes serious genotoxicity and thus requires strict processing condition for minimization of human exposure.Diverse methods including incineration,absorption,oxidation,condensation and biodegradation have been developed for removal of DCM from environments.Among them,biological methods gain increasing popularity due to their mild reaction conditions,low cost,high removal rate and no secondary pollution.DCM dehalogenase in microbial cells can catalyze the degradation of deleterious DCM in environments.However,the utility of naturally occurring DCM dehalogenase in microbial cells is often limited due to slow mass transfer and low enzyme activity and yield.This study aims to improve the expression of DCM dehalogenase by heterologous expression and optimizing the conditions of enzyme production.In order to obtain DCM dehalogenase with higher activity,the structure of DCM dehalogenase was explored and modified by bioinformatics and related experiments.First,the gene encoding DCM dehalogenase was cloned from Methylobacterium rhodesianum and overexpressed in Escherichia coli.The enzyme production conditions of the recombinant strain were optimized,and we found that the optimal production conditions are 20? of induction temperature,8 hours of induction time and 7 of pH.The molecular chaperone co-expressing strain was constructed and the protein expressed by the chaperone plasmid pGro7 was found to increase the expression of soluble DCM dehalogenase by nearly 50%.By folding renaturation of inclusion bodies,we obtained more soluble DCM dehalogenase.Then,the structure of DCM dehalogenase was predicted using homology modeling and conserved domain analysis.It was found that DCM dehalogenase belongs to the glutathione transferase(GST)family which has an N-terminal ?-sheet domain and a C-terminal ?-helix domain.The substrate located in the fissure between the Cterminal alpha helices.Based on molecular docking analysis of DCM dehalogenase using DCM as the ligand,all of the target amino acid residues within substrate binding pocket and 10 conservative amino acid residues were individually mutated to alanine(Ala).After determination of activity,R120,L121,W128 and T146 were chosen for saturation mutation.Results showed that dcmT146 A,dcmT146R and dcmT146 Q have higher activities,whereas dcmL121 A,dcmT146L,dcmL121 Q and dcmL121 F have retained activities.Next,these seven mutants with single mutation on amino acid residue were chosen for double mutation.It was found that the mutant of dcmL121A/T146 R exhibits the highest activity increasing by 52.8% relative to wild type.The relationship between structure and function was explored by analyzing the structure of wild enzyme and mutant dcmL121 A / T146 R.And we found that the mutant variant dcmL121A/T146 R bears the reduced steric hindrance in active center with decreased number of amino acid residues within binding pocket from 8 to 5 while increased overall hydrophilicity.In addition,the number of hydrophobic amino acid residues within substrate binding pocket increased while Km value decreased.It was speculated that all these changes in mutant variant dcmL121A/T146 R may contribute to the increase in catalytic activity.In addition,the enzymatic properties of the two were compared and found that the mutant of dcmL121A/T146 A has overall similar change pattern in activity as wild type.In order to improve the service life of DCM dehalogenase,we carried out immobilization studies on enzymes.We use graphene oxide(GO)to immobilize the enzyme and improve the thermal stability of DCM dehalogenase.The study also found that under the action of the cross-linking agent and metal ion,the immobilization efficiency of GO is greatly improved.The optimum pH of the immobilized enzyme shifts to alkaline.After 6 times of immobilized enzyme use,the enzyme activity remained at about 50%. |
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