title: Expression, Purification and Characterisation of Secreted Esterase Rv2525c from Mycobacterium tuberculosis.
abstract: 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 ≤ 40 °C. Moreover, we tested the esterase activity using p-nitrophenyl esters (C2, C4, C6, C8, C12, C14, C16). This enzyme exhibited broad substrate acceptance, preferentially hydrolysing p-nitrophenyl butyrate (C4) at pH 7.0 and 37 °C. The dynamic activity test demonstrated that the optimal conditions were pH 8.0 and 38 °C. Site-directed mutagenesis studies revealed that Gly 113, Ser 115 and Gly 117 residues play catalytic roles in Rv2525c.
authors: Division of Bacterial Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 427 Maduan Street, Nangang Dist., Harbin, 15000, People's Republic of China.
authors: Guanghui
authors: G
authors: Dang
authors: Liping
authors: L
authors: Chen
authors: Zhaoli
authors: Z
authors: Li
authors: Xiaoxia
authors: X
authors: Deng
authors: Yingying
authors: Y
authors: Cui
authors: Jun
authors: J
authors: Cao
authors: Shenye
authors: S
authors: Yu
authors: Hai
authors: H
authors: Pang
authors: Siguo
authors: S
authors: Liu
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004950:Esterases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D015870:Gene Expression; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D004950:Esterases