A novel esterase from Ralstonia sp. M1: Gene cloning, sequencing, high-level expression and characterization

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• 作者：Dinh Thi Quyen ; Thi Tuyet Dao ; Sy Le Thanh Nguyen
• 关键词：Ralstonia sp. M1 ; Esterase gene ; Cloning ; Expression ; Characterization
• 刊名：Protein Expression and Purification
• 出版年：2007
• 期刊代码：184_10465928
• 类别：bio
• 出版时间：February 2007
• 卷：51
• 期：2
• 页码：133-140
• 文件大小：1172 K

摘要

A newly isolated gene from Ralstonia sp. M1, encoding an esterase, was cloned in Escherichia coli and its nucleotide sequence determined. The 1.6 kb insert revealed one complete open reading frame, predicted to encode an esterase (320 aa, 34.1 kDa) with a pI of 9.86. EstR contained a putative oxyanion hole H³⁶G³⁷, a conserved pentapeptide G¹⁰³HSLG¹⁰⁷ and a conserved catalytic His²⁶⁵ and Asp²³⁷. The EstR sequence shared 64–70 and 44–48%identity with the hydrolases/acyltransferases from Burkholderia strains and from Ralstonia strains, respectively, 44 and 38%identity with the lactone-specific esterase from Pseudomonas fluorescens and Mesorhizobium loti, respectively. The esterase EstR was expressed with a high level of 41 mg/g wet cells. The Ni–NTA-purified esterase EstR showed an optimal activity in the temperature range 60–65 °C and pH range 7.5–9.0 towards p-nitrophenyl caproate. The enzyme was found to be highly resistant to many organic solvents especially induced by ethanolamine. Metal ions showed slight effect on esterase activity. The inhibitor phenylmethanesulfonyl fluoride inhibited strongly the esterase. Triton X-45 induced the activation of EstR, but other detergents slightly to strongly decreased or completely inhibited. Among tested p-NP esters, caproate was the most preferential substrate of this esterase.