Molecular Docking and Site-Directed Mutagenesis of Dichloromethane Dehalogenase to Improve Enzyme Activity for Dichloromethane Degradation
Abstract
Dichloromethane (DCM) dehalogenase in bacterial cells can catalyze the degradation of deleterious DCM in environments. However, the utility of naturally occurring DCM dehalogenase is often limited due to low enzyme activity and content in living cells. In this study, the gene encoding DCM dehalogenase was cloned from Methylobacterium rhodesianum and overexpressed in Escherichia coli. 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 Ala. After determination of activity, R120, L121, W128, and T146 were chosen for further saturation mutation. Results showed that dcmT146A, dcmT146R, and dcmT146Q have higher activities, whereas dcmL121A, dcmT146L, dcmL121Q, and dcmL121F have retained activities. Next, these seven mutants with a single mutation on amino acid residue were chosen for double mutation. It was found that the mutant of dcmL121A/T146R exhibits the highest activity increasing by 52.8% relative to wild type. Bioinformatic and experimental analyses revealed that the mutant variant dcmL121A/T146R bears the reduced steric hindrance in the active center with a d...Continue Reading
References
Effective utilization of dichloromethane by a newly isolated strain Methylobacterium rhodesianum H13
Structural Analysis and Aggregation Propensity of Pyroglutamate Aβ(3-40) in Aqueous Trifluoroethanol
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