Cloning and characterization of a thermostable carboxylesterase from inshore hot spring thermophile Geobacillus sp. ZH1

• Published in: Acta oceanologica Sinica Vol. 31; no. 6; pp. 117 - 126
• Main Authors: Zhu, Yanbing, Liu, Guangming, Li, Hebin, Liu, Jingwen, Bai, Xiaoming, Guan, Rong, Cai, Huinong
• Format: Journal Article
• Language: English
• Published: Heidelberg The Chinese Society of Oceanography 01.11.2012; Springer Nature B.V
• Subjects: Acetates; Acetic acid; Carboxylesterase; Climatology; Cloning; Detergents; E coli; Earth and Environmental Science; Earth Sciences; Ecology; Engineering Fluid Dynamics; Environmental Chemistry; Enzymatic activity; Enzyme activity; Enzymes; Escherichia coli; Esterase; Esterases; Esters; Fluorides; Geobacillus; Hot springs; Industrial applications; Marine; Marine & Freshwater Sciences; Microorganisms; Molecular weight; Oceanography; Organic solvents; p-Nitrophenyl butyrate; p-Nitrophenylacetic acid; Phenylmethylsulfonyl fluoride; Recombinants; SDS-PAGE分析; Serine; Solvents; Structural analysis; Substrates; Thermophilic bacteria; Xylene; 克隆; 嗜热芽孢杆菌; 对硝基苯基; 羧酸酯酶; 耐热性; 酯酶活性; 鉴定; Fujian China; China; carboxylesterase; characterization; expression
• ISSN: 0253-505X; 1869-1099
• DOI: 10.1007/s13131-012-0258-0

The gene （741 bp） encoding carboxylesterase from the thermophilic bacterium Geobacillus sp. ZH1 was cloned and overexpressed in Escherichia coll. The purified recombinant protein presented a molecular mass of about 40 kDa by SDS-PAGE analysis. Enzyme assays using p-nitrophenyl esters with different acyl chain lengths as the substrates confirmed its esterase activity, yielding highest specific activity with p-nitrophenyl acetate. Among the p-nitrophenyl esters tested, the carboxylesterase presented preference for p-nitrophenyl caprylate, but hydrolyzed p-nitrophenyl butyrate more efficiently. When p-nitrophenyl butyrate was used as a substrate, the recombinant carboxylesterase exhibited highest activity at pH 8.0 and 60℃. Almost no decrease in esterase activity was observed at 60℃ for 3 h, and over 40% of activity was still maintained after incubation at 90℃ for 3 h. These results indicate that Geobacillus sp. ZH1 recombinant esterase was thermostable. The enzymatic activity was inhibited by the addition of phenylmethylsulfonyl fluoride, indicating that it contains serine residue, which plays a key role in the catalytic mechanism. Except SDS and xylene, this esterase showed stability toward other tested detergents and organic solvents. Cloning, expression, and biochemical characterization of Geobacillus sp. ZH1 carboxylesterase lay a good foundation for its structural characterization and industrial application.