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Journal of Bacteriology, December 2008, p. 8086-8095, Vol. 190, No. 24
0021-9193/08/$08.00+0     doi:10.1128/JB.00803-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

A Novel Thermostable Arylesterase from the Archaeon Sulfolobus solfataricus P1: Purification, Characterization, and Expression{triangledown} ,{dagger}

Young-Jun Park, Sung-Jin Yoon, and Hee-Bong Lee*

Department of Biochemistry, College of Natural Sciences, Kangwon National University, Chuncheon 200-701, South Korea

Received 8 June 2008/ Accepted 2 October 2008

A novel thermostable arylesterase, a 35-kDa monomeric enzyme, was purified from the thermoacidophilic archaeon Sulfolobus solfataricus P1. The optimum temperature and pH were 94°C and 7.0, respectively. The enzyme displayed remarkable thermostability: it retained 52% of its activity after 50 h of incubation at 90°C. In addition, the purified enzyme showed high stability against denaturing agents, including various detergents, urea, and organic solvents. The enzyme has broad substrate specificity besides showing an arylesterase activity toward aromatic esters: it exhibits not only carboxylesterase activity toward tributyrin and p-nitrophenyl esters containing unsubstituted fatty acids from butyrate (C4) to palmitate (C16), but also paraoxonase activity toward organophosphates such as p-nitrophenylphosphate, paraoxon, and methylparaoxon. The kcat/Km ratios of the enzyme for phenyl acetate and paraoxon, the two most preferable substrates among all tested, were 30.6 and 119.4 s–1·µM–1, respectively. The arylesterase gene consists of 918 bp corresponding to 306 amino acid residues. The deduced amino acid sequence shares 34% identity with that of arylesterase from Acinetobacter sp. strain ADP1. Furthermore, we successfully expressed active recombinant S. solfataricus arylesterase in Escherichia coli. Together, our results show that the enzyme is a serine esterase belonging to the A-esterases and contains a catalytic triad composed of Ser156, Asp251, and His281 in the active site.

* Corresponding author. Mailing address: Department of Biochemistry, College of Natural Sciences, Kangwon National University, Chuncheon 200-701, South Korea. Phone: 82-33-250-8512. Fax: 82-33-242-0459. E-mail: leehbong{at}kangwon.ac.kr

{triangledown} Published ahead of print on 17 October 2008.

{dagger} Supplemental material for this article may be found at http://jb.asm.org/.

Journal of Bacteriology, December 2008, p. 8086-8095, Vol. 190, No. 24
0021-9193/08/$08.00+0     doi:10.1128/JB.00803-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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