Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of life sciences, Nanjing Agricultural University, Nanjing, 210095, PR China.
Industrial Product Division, Intrexon Corporation, South San Francisco, CA, 94080, USA.
J Hazard Mater. 2017 Jun 5;331:55-62. doi: 10.1016/j.jhazmat.2017.02.007. Epub 2017 Feb 16.
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 MBC at the average degradation rate of 0.63mgLh. 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.
一株新型的多菌灵（甲基-1H-苯并咪唑-2-基氨基甲酸酯，或 MBC）降解菌株 SD-4 被分离并初步鉴定为分枝杆菌属。根据其表型特征和系统发育分析。该菌株可以利用 MBC 作为唯一的碳源和氮源进行生长，并以平均降解速率 0.63mgLh 降解 50mgL 的 MBC。菌株 SD-4 通过典型途径降解 MBC，其中 MBC 首先被 MheI 水解为 2-氨基苯并咪唑（2-AB），然后转化为 2-羟基苯并咪唑（2-HB）。MBC 水解酶编码基因 mheI 从菌株 SD-4 中克隆，并通过密码子优化成功在大肠杆菌中表达。巯基阻断试验表明，MheI 的活性与半胱氨酸密切相关，定点突变实验表明，Cys16 和 Cys222 在 MheI 水解 MBC 过程中发挥重要作用。因此，它们直接影响其活性，被定义为关键氨基酸位点。
