| ZHU Yanbing,LIU Guangming,LI Hebin,LIU Jingwen,BAI Xiaoming,GUAN Rong,CAI Huinong. 2012. Cloning and characterization of a thermostable carboxylesterase from inshore hot spring thermophile Geobacillus sp. ZH1. Acta Oceanologica Sinica, (6):117－126 |
| Cloning and characterization of a thermostable carboxylesterase from inshore hot spring thermophile Geobacillus sp. ZH1 |
| Cloning and characterization of a thermostable carboxylesterase from inshore hot spring thermophile Geobacillus sp. ZH1 |
| Received:July 27, 2011 Revised:February 25, 2012 |
| DOI：10.1007/s13131-012-0258-0 |
| Key words:carboxylesterase expression characterization |
| 中文关键词: carboxylesterase expression characterization |
| 基金项目:Scientific Research Fund of Fujian Provincial Education Department, China under contact No. JA11153; the Natural Science Foundation of Fujian Province, China under contact Nos 2010J06012 and 2010J01261; the Foundation for Innovative Research Team of Jimei University, China under contact No. 2010A005. |
| Author Name | Affiliation | E-mail | | ZHU Yanbing | School of Biotechnology Engineering, Jimei University, Xiamen 361021, China
Research Center of Food Microbiology and Enzyme Engineering Technology(Jimei University), Fujian Province University, Xiamen 361021, China
Research Center of Food Biotechnology of Xiamen City, Xiamen 361021, China | | | LIU Guangming | School of Biotechnology Engineering, Jimei University, Xiamen 361021, China | | | LI Hebin | Xiamen Medical College, Xiamen 361008, China | | | LIU Jingwen | School of Biotechnology Engineering, Jimei University, Xiamen 361021, China | | | BAI Xiaoming | School of Biotechnology Engineering, Jimei University, Xiamen 361021, China | | | GUAN Rong | School of Biotechnology Engineering, Jimei University, Xiamen 361021, China | | | CAI Huinong | School of Biotechnology Engineering, Jimei University, Xiamen 361021, China
Research Center of Food Microbiology and Enzyme Engineering Technology(Jimei University), Fujian Province University, Xiamen 361021, China
Research Center of Food Biotechnology of Xiamen City, Xiamen 361021, China | jmswthermo@hotmail.com |
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| Abstract: |
| The gene (741 bp) encoding carboxylesterase from the thermophilic bacterium Geobacillus sp. ZHl was cloned and overexpressed in Escherichia coli. 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. |
| 中文摘要: |
| The gene (741 bp) encoding carboxylesterase from the thermophilic bacterium Geobacillus sp. ZHl was cloned and overexpressed in Escherichia coli. 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. |
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