Hydrolysis mechanism of carbendazim hydrolase from the strain Microbacterium sp. djl-6F
Ji Lei
,
Lijun Ren
,
Peng Chen
,
Shaopeng Wei
,
Shibin Hu
DOI:10.1016/j.jes.2016.05.027
Received March 25, 2016,Revised May 10, 2016, Accepted May 26, 2016, Available online July 09, 2016
Volume 29,2017,Pages 171-177
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 turnover 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.
