烟草肠杆菌芳香族氨基酸转氨酶的结构预测、克隆表达及酶学性质研究
Structural prediction, cloning, expression and enzymatic propertity of aromatic amino acid transaminase from Enterobacter tabaci
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摘要: 为提升芳香族氨基酸转氨酶(AAT)在合成生长素、促进作物生长方面的潜在应用价值,采用生物信息学方法对烟草肠杆菌β7(Enterobacter tabaci strain β7) AAT的结构进行分析和预测,利用大肠杆菌对该酶进行异源克隆表达和纯化,并测定重组AAT的酶学性质。结果表明:AAT含有396个氨基酸,该酶活性中心关键氨基酸残基为Lys246;由大肠杆菌异源表达的重组AAT的分子质量约为43 kDa,其最适底物为色氨酸,最适反应温度为50℃、pH值为8.0;重组AAT对乙醇具有较好的耐受性,且Cu2+对该酶活性有一定的促进作用。该研究结果可为烟草肠杆菌AAT的酶分子改造和高产生长素工程菌的构建提供理论基础。Abstract: To enhance the potential application of aromatic amino acid transaminase (AAT) in the synthesis of auxin (IAA) and promotion of crop growth, bioinformatics methods were applied to analyze and predict the structure of AAT from Enterobacter tabaci strain β7. Then, this AAT was cloned, expressed and purified in Escherichia coli, and the enzymatic properties of recombinant AAT were determined. The bioinformatics analysis results revealed that the length of this AAT protein is composed of 396 amino acids. The key residue in the active center of this AAT was Lys246. The molecular weight of the recombinant AAT expressed in E.coli was approximately 43 kDa. The optimal substrate for recombinant AAT was tryptophan, at a temperature of 50 ℃ and a pH value of 8.0. Cu2+ had a certain activating effect on enzyme activity, and recombinant AAT showed good tolerance to the organic solvent ethanol. These results provided a theoretical basis for the AAT enzyme modification and the construction of high-yield auxin engineering bacteria.
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