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The carbendazim (MBC) hydrolyzing enzyme gene was cloned and heterologously expressed in Escherichia coli BL21 (DE3) from a newly isolated MBC-degrading bacterium strain Microbacterium sp.strain djl-6F.High performance liquid chromatography-mass spectrometry (HPLC-MS) analysis revealed that purified MheI-6F protein catalyzes direct hydrolysis of MBC into 2-aminobenzimidazole (2-AB) with a high tover rate and moderate affinity (Km of 6.69 μmol/L and kcat of 160.88/min) without the need for any cofactors.The optimal catalytic condition of MheI-6F was identified as 45℃C,pH 7.0.The enzymatic activity of MheI-6F was found to be diminished by metal ions,and strongly inhibited by sodium dodecyl sulfate (SDS).Through generating amino acid mutations in MheI-6F,Cys16 and Cys222 were identified as the catalytic groups that are essential for the hydrolysis of MBC.This is the first report on the biodegradation of MBC at the enzymatice level.