Identification of the key amino acid sites of the carbendazim hydrolase (MheI) from a novel carbendazim-degrading strain Mycobacterium sp. SD-4

• Published in: Journal of hazardous materials Vol. 331; pp. 55 - 62
• Main Authors: Zhang, Yingkun, Wang, Hui, Wang, Xiang, Hu, Bo, Zhang, Chenfei, Jin, Wen, Zhu, Shijun, Hu, Gang, Hong, Qing
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 05.06.2017
• Subjects: Benzimidazoles - metabolism; Carbamates - metabolism; Carbendazim; Escherichia coli; Expression; Genes, Bacterial; Hydrolases - chemistry; Hydrolases - genetics; Hydrolases - metabolism; Key amino acid sites; mheI; Mycobacterium - enzymology; Mycobacterium - genetics; Mycobacterium - isolation & purification; Mycobacterium sp. SD-4; mheI; Mycobacterium sp. SD-4; Expression; Carbendazim; Key amino acid sites
• ISSN: 0304-3894; 1873-3336
• DOI: 10.1016/j.jhazmat.2017.02.007

•Strain SD-4 was the first MBC-degrading strain isolated from the genus of Mycobacterium.•The codon of mheI was optimized to realize its soluble expression in E.coil.•Key amino acid sites of MheI were identified. A novel carbendazim (methyl-1H-benzimidazol-2-ylcarbamate, or MBC) degrading strain SD-4 was isolated and identified preliminarily as Mycobacterium sp. according to its phenotypic features and phylogenetic analysis. This strain could utilize MBC as the sole carbon and nitrogen sources for growth and degrade 50mgL−1 MBC at the average degradation rate of 0.63mgL−1h−1. Strain SD-4 degraded MBC through the typical pathway, in which MBC was first hydrolyzed by MheI to 2-aminobenzimidazole (2-AB) and then converted to 2-hydroxybenzimidazole (2-HB). The MBC hydrolase encoding gene mheI was cloned from strain SD-4 and successfully expressed in Escherichia coli by codon optimization. The sulfhydryl-blocking assay revealed that the activity of MheI was closely related to cysteine, and the site-directed mutation experiment showed that Cys16 and Cys222 played important roles during the hydrolysis of MBC by MheI. Therefore they affected its activity directly and were defined as the key amino acid sites.