Expression, Purification and Characterisation of Secreted Esterase Rv2525c from Mycobacterium tuberculosis

文献类型： 外文期刊

第一作者： Dang, Guanghui

作者： Dang, Guanghui;Chen, Liping;Li, Zhaoli;Cui, Yingying;Cao, Jun;Yu, Shenye;Liu, Siguo;Deng, Xiaoxia;Pang, Hai

作者机构：

关键词： Mycobacterium tuberculosis;Secreted esterase;Rv2525c

期刊名称：APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY （ 影响因子：2.926； 五年影响因子：2.685 ）

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收录情况： SCI

摘要： Rv2525c from Mycobacterium tuberculosis belongs to the domain of unknown function (DUF) 1906 superfamily, but it also contains the motif G-X-S-X-G, the consensus active site sequence of the ester/lipid family. Biochemical analysis indicated that the mature Rv2525c protein is secreted. The discovery and characterisation of novel enzymes secreted by M. tuberculosis are vital for understanding the pathogenesis of the most important human bacterial pathogen. The proteome of M. tuberculosis contains over 400 potentially secreted proteins, of which the majority remain uncharacterised. In this study, we cloned and expressed the rv2525c gene in Escherichia coli and purified the recombinant protein using a three-step process (affinity chromatography, ion exchange chromatography, gel filtration chromatography), obtaining more than 99 % pure protein. Mass spectrometry was performed to confirm that the purified protein was Rv2525c. Circular dichroism spectroscopy results showed that its conformation was stable at pH ranging from 6.0 to 8.0 and at temperatures a parts per thousand currency sign40 A degrees C. Moreover, we tested the esterase activity using p-nitrophenyl esters (C-2, C-4, C-6, C-8, C-12, C-14, C-16). This enzyme exhibited broad substrate acceptance, preferentially hydrolysing p-nitrophenyl butyrate (C-4) at pH 7.0 and 37 A degrees C. The dynamic activity test demonstrated that the optimal conditions were pH 8.0 and 38 A degrees C. Site-directed mutagenesis studies revealed that Gly 113, Ser 115 and Gly 117 residues play catalytic roles in Rv2525c.

分类号： Q5

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