Cloning And Characterization Of Dichloromethane Dehalogenase For Dichloromethane Degradation

Dichloromethane(DCM) is a widely used industrial solvent and a known carcinogen. It causes serious environmental pollution and becomes a focus of environmental governance. Traditional microbial degradation of DCM has several drawbacks. First, the degradation time is very longe; second, the process is easy to be contaminated by other microorganisms; third, it is difficult to control the process. To avoid these problems, we are going to clone and express DCM dehalogenase from M. rhodesianum in E. coli. After purification, the pure DCM dehalogenase was used to degrade DCM. Use of pure DCM dehalogenase for DCM degradation gives the following advantages: Efficiency and specificity are high; enzymatic condition is simple; reaction is quick; condition is easy to control.The complete open reading frame(ORF) of the DCM dehalogenase gene(access no. KM243642) in M. rhodesianum was predicted to encode a polypeptide containing 289 amino acids with formula of C2511H4157N867O1043S210. The putative molecular weight and theoretical isoelectricpoint(PI) were 269.00 for ssDNA or 534.55 for dsDNA and 6.33, respectively. There were 217 hydrophobic amino acids(A, I, L, F, W, V, P, M) and 650 hydrophilic amino acids(G, S, T, N, C, Q, Y, D, E, R, K, H). Compared with DCM dehalogenase gene from Methylobacterium extorquens DM4, the one from M. rhodesianum has only one difference, that C from Methylobacterium extorquens DM4 was replaced by G M. rhodesianum at 62 bp.DCM dehalogenase was subsequently expressed in Escherichia coli BL21(DE3) and purified. It was found that the enzyme activity in recombinant cells was three times higher than the one in wild type M. rhodesianum. Further investigation showed that the recombinant DCM dehalogenase was the most active at 40°C under pH 7-8 and its Km was 10.96 mM when treated with DCM as substrate. During the degradation of DCM by the recombinant DCM dehalogenase, HCl and formaldehyde were detected, thus for the first time verifying the postulated GSH-involved reaction mechanism.From 0 to 135 min during degradation, the concentration of chloride ions increased from 7.477mg/l to 34.376mg/l, and the degradation rate reached 0.479mM/h. Compared with Methylotrophic bacterium DM1 and Hyphomicrobium DM2, the degradation rate of methylene chloride improves 1.6-fold and 2-fold, respectively. Compared to conventional microbiological degradation, DCM degradation by using pure enzyme gave higher degradation efficiency in short time, the process is easier to control, and there is lower risk of contamination.