Identification of the key amino acid sites of the carbendazim hydrolase (MheI) from a novel carbendazim-degrading strain Mycobacterium sp. SD-4.
Identification of the key amino acid sites of the carbendazim hydrolase (MheI) from a novel carbendazim-degrading strain Mycobacterium sp. SD-4.
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DOI：
10.1016/j.jhazmat.2017.02.007
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发表时间：
2017-06
影响因子：
13.6
通讯作者：
Yingkun Zhang;Hui Wang;Xiang Wang;Bo Hu;Chenfei Zhang;Wen Jin;Shijun Zhu;Gang Hu;Q. Hong
中科院分区：
文献类型：
--
作者：
Yingkun Zhang;Hui Wang;Xiang Wang;Bo Hu;Chenfei Zhang;Wen Jin;Shijun Zhu;Gang Hu;Q. Hong
分离到一株新的多菌灵降解菌SD-4，经初步鉴定为分枝杆菌属（Mycobacteriumsp.）根据其表型特征和系统发育分析，该菌株能以MBC为唯一碳源和氮源生长，降解50 mg L-1 MBC，平均降解速率为0.63 mg L-1h-1。菌株SD-4降解MBC的典型途径为：MBC先被MheI水解为2-氨基苯并咪唑（2-AB），然后再转化为2-羟基苯并咪唑（2-HB）。从SD-4菌株中克隆了MBC水解酶基因heI，并通过密码子优化在大肠杆菌中成功表达。巯基阻断实验表明MheI的活性与半胱氨酸密切相关，定点突变实验表明Cys 16和Cys 222在MheI水解MBC过程中起重要作用。因此，它们直接影响其活性，并被定义为关键氨基酸位点。
A novel carbendazim (methyl-1H-benzimidazol-2-ylcarbamate, or MBC) degrading strain SD-4 was isolated and identified preliminarily asMycobacteriumsp. according to its phenotypic features and phylogenetic analysis. This strain could utilize MBC as the sole carbon and nitrogen sources for growth and degrade 50 mg L−1MBC at the average degradation rate of 0.63 mg L−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 genemheIwas cloned from strain SD-4 and successfully expressed inEscherichia coliby 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.