A carboxylesterase from the thermoacidophilic archaeon Sulfolobus solfataricus P1; purification,characterization, and expression |
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Authors: | Young-Jun Park Soo Young Choi Hee-Bong Lee |
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Institution: | 1. Department of Biochemistry, College of Natural Sciences, Kangwon National University, Chuncheon 200-701, Republic of Korea;2. Department of Biomedical Sciences and Institute for Bioscience and Biotechnology, Hallym University, Chuncheon 200-702, Republic of Korea |
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Abstract: | The carboxylesterase, a 34 kDa monomeric enzyme, was purified from the thermoacidophilic archaeon Sulfolobus solfataricus P1. The optimum temperature and pH were 85 °C and 8.0, respectively. The enzyme showed remarkable thermostability: 41% of its activity remained after 5 days of incubation at 80 °C. In addition, the purified enzyme exhibited stability against denaturing agents, including various detergents, urea, and organic solvents. The enzyme has broad substrate specificity towards various PNP esters and short acyl chain triacylglycerols such as tributyrin (C4:0). Among the PNP esters tested, the best substrate was PNP-caprylate (C8) with Km and kcat values of 71 μM and 14,700 s−1, respectively. The carboxylesterase gene consisted of 915 bp corresponding to 305 amino acid residues. We demonstrated that active recombinant S. solfataricus carboxylesterase could be expressed in Escherichia coli. The enzyme was identified as a serine esterase belonging to mammalian hormone-sensitive lipases (HSL) family and contained a catalytic triad composed of serine, histidine, and aspartic acid in the active site. |
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Keywords: | Carboxylesterase Thermostability Chemostability Archaea Sulfolobus solfataricus |
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