一株MAs (III)脱甲基菌株的分离及功能基因的研究

Identification of an methylarsenite demethylation bacterium Bacillus aryabhattai FJ-6 and characterization of methylarsenite demethylation gene

摘要:

[目的] 分离并鉴定三价单甲基砷（MAs （III））脱甲基菌株，对MAs （III）脱甲基菌FJ-6中arsI基因进行克隆表达，并对arsI基因表达蛋白进行功能鉴定。[方法] 利用富集培养的方法分离MAs （III）脱甲基菌株，并通过形态学、生理生化特征和16S rDNA基因进化分析进行鉴定；HPLC-ICP-MS鉴定菌株转化MAs （III）的产物为三价砷（As （III）），对菌株FJ-6的基因组进行生物信息学分析，寻找潜在的MAs （III）脱甲基酶编码基因，通过PCR扩增获得arsI全长基因，构建重组质粒pET29a-arsI，转化大肠杆菌BL21（DE3）菌株进行异源表达，通过Ni²⁺-NTA亲和层析柱纯化异源表达的蛋白，以MAs （III）为反应底物，检测MAs （III）脱甲基酶ArsI的酶学特性。通过实时定量PCR观察arsI的表达类型。[结果] Bacillus aryabhattai FJ-6在12 h内能将1 μmol/L MAs （III）完全转化为As （III）。克隆得到MAs （III）脱甲基酶表达基因arsI，构建了pET29a-arsI重组质粒并进行了表达，ArsI蛋白分子量为17.4 kDa。ArsI纯化蛋白具有较高的MAs （III）脱甲基酶的活性；荧光定量PCR实验结果表明arsI受砷诱导表达。[结论] 明确了ArsI蛋白具有MAs （III）脱甲基酶活性。

Abstract:

[Objective] The purpose of this study is to isolate and identify the MAs(III) demethylation strains. We cloned and expressed the arsI gene involved in MAs(III)-demethylation in Bacillus aryabhattai FJ-6 and characterized the ArsI protein.[Methods] The arsI gene fragment (432 bp) was amplified with PCR. Recombinant plasmid pET29a-arsI was constructed and transformed into Escherichia coli BL21(DE3) for heterologous expression, and the expressed protein was detected with SDS-PAGE. The enzyme activity of ArsI was determined by using HPLC-ICP-MS.[Results] The arsI gene was cloned and the recombinant plasmid pET29a-arsI was expressed. The molecular weight of the recombinant protein was 17.4 kDa.[Conclusion]] The activity of ArsI protein with MAs(III) demethylation was clarified.