A new method was used to preparing genomic DNA from Microbacterium sp.quickly and efficiently.DNA quantity and purity was measured by UV absorbance.Integrity of the genomic DNA was tested by agarose gel eletrophoresis...A new method was used to preparing genomic DNA from Microbacterium sp.quickly and efficiently.DNA quantity and purity was measured by UV absorbance.Integrity of the genomic DNA was tested by agarose gel eletrophoresis.The DNA prepared by this method was sufficiently pure for PCR.This method saves time and cost,practices easily as well.展开更多
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 perfor...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 into2-aminobenzimidazole（2-AB） with a high turnover rate and moderate affinity（Kmof6.69 μmol/L and kcatof 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.展开更多
文摘A new method was used to preparing genomic DNA from Microbacterium sp.quickly and efficiently.DNA quantity and purity was measured by UV absorbance.Integrity of the genomic DNA was tested by agarose gel eletrophoresis.The DNA prepared by this method was sufficiently pure for PCR.This method saves time and cost,practices easily as well.
基金supported by the Hi-Tech Research and Development Program(863) of China(No.2012AA101404)the National Natural Science Foundation of China(No.31301700)+1 种基金the Science and Technology Program of BaoJi(No.2013R6-3)the Key Subject Project of Baoji University of Arts and Sciences(No.ZK0919)
文摘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 into2-aminobenzimidazole（2-AB） with a high turnover rate and moderate affinity（Kmof6.69 μmol/L and kcatof 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.
