The Bioelectrocatalytic Dechlorination Of Dichloromethane At The Dehalogenases On Multiwalled Carbon Nanotubes Modified Graphite Electrode

The dichloromethane?DCM?bioelectrochemical dechlorination has important academic significance.In this work,the DCM dehalogenase gene?dcm?has been cloned from the Methylobacterium rhodesianum H13 and the prokaryotic expression recombinant bacteria E.coil BL21-pET28b?+?-dcm has been constructed for getting the DCM dehalogenase.On the base of that,we obtained more biological information of the DCM dehalogenase by bioinformatics analysis which is very useful for furture research.Affinity chromatography combined with ultrafiltration was used to get enzyme of higher purity,and a coupled assay with NAD-dependent formaldehyde dehydrogenase from Pseudomonas putida was used for determining the specific activity of DCM dehalogenase.A sodium alginate/DCM dehalogenase-multiwalled carbon nanotubes-graphite composite electrode?SA/De-MWCNT-GE?were prepared with gel immobilized method,and the catalytic activity of the cathodes as well as the pathway of DCM on them were investigated.The main scientific findings are as follows:?1?Compared with the dcm from Methylobacterium.extorquens DM4,the cloned gene replaced cytosine with guanine at the 62nd base.It was found to be 864bp and encoded 288 amino acids with a molecular weight of 33 kDa.?2?The DCM dehalogenase(C₁₅₀₅H₂₂₅₅N₄₀₃O₄₃₀S₇)was a stable hydrophilic proteins in the cytoplasm with a pI of 6.35.DCM dehalogenase was a curly hair protein with 52.43%of the amino acid residues exposed on its surface,and it contains53.47%alpha-helix which was the maximum structural components.As one of the theta class glutathione transferases,DCM dehalogenase was in the form of dimer and showed even stronger substrate selectivity.The catalytic channel radius and length of DCM dehalogenase were both 1.4?.Glu42 and Val43 were two of the essential amino acids in both the catalytic channel and the catalytic pockets.The amino acid sequence of DCM dehalogenase predicted to be homologous among the microorganism,and the active center was conservative with 8 potential sites.?3?DCM dehalogenase was an endoenzyme which was highly expressed in soluble form after inducted by IPTG in E.coil BL21-pET28b?+?-dcm,and its molecular weight was corresponds to the predicted value.After purified by the method of affinity chromatography combined with ultrafiltration,the specific activity of the DCM dehalogenase was 0.03647±0.00084 U/mg,which was 2 times and 7.4times higher than that purified by only affinity chromatography and the crude enzyme,respectively.Glycerin-cryopreserved at-20?was confirmed to be a safe way for the pure enzyme activity-preserving storage in a long-term.?4?The removal efficiency and average current efficiency of DCM dechloridation was 49.2%and 79.4%respectively after 20 hours electrolysis with SA/De-MWCNT-GE in pH 7 PBS at 298 K while the DCM initial concentration was10 mM.The total mass balance of the reactant has been detected,and the products were in the range of 98-102%during the bioelectrocatalytic reductive reaction.Based on the intermediates detected,a pathway was proposed for DCM degradation in which it underwent dechlorination process.