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Journal of Bacteriology, May 2009, p. 3076-3085, Vol. 191, No. 9
0021-9193/09/$08.00+0     doi:10.1128/JB.01060-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Characterization of a Novel Thermostable Carboxylesterase from Geobacillus kaustophilus HTA426 Shows the Existence of a New Carboxylesterase Family{triangledown}

Silvia Montoro-García,1 Irene Martínez-Martínez,1 José Navarro-Fernández,1 Hideto Takami,2 Francisco García-Carmona,1 and Álvaro Sánchez-Ferrer1*

Department of Biochemistry and Molecular Biology-A, Faculty of Biology, University of Murcia, Campus Espinardo, E-30100 Murcia, Spain,1 Microbial Genome Research Group, Japan Agency for Marine-Earth Science and Technology, 2-15 Natsushima, Yokosuka 237-0061, Japan2

Received 30 July 2008/ Accepted 18 February 2009

The gene GK3045 (741 bp) from Geobacillus kaustophilus HTA426 was cloned, sequenced, and overexpressed into Escherichia coli Rosetta (DE3). The deduced protein was a 30-kDa monomeric esterase with high homology to carboxylesterases from Geobacillus thermoleovorans NY (99% identity) and Geobacillus stearothermophilus (97% identity). This protein suffered a proteolytic cut in E. coli, and the problem was overcome by introducing a mutation in the gene (K212R) without affecting the activity. The resulting Est30 showed remarkable thermostability at 65°C, above the optimum growth temperature of G. kaustophilus HTA426. The optimum pH of the enzyme was 8.0. In addition, the purified enzyme exhibited stability against denaturing agents, like organic solvents, detergents, and urea. The protein catalyzed the hydrolysis of p-nitrophenyl esters of different acyl chain lengths, confirming the esterase activity. The sequence analysis showed that the protein contains a catalytic triad formed by Ser93, Asp192, and His222, and the Ser of the active site is located in the conserved motif Gly91-X-Ser93-X-Gly95 included in most esterases and lipases. However, this carboxylesterase showed no more than 17% sequence identity with the closest members in the eight families of microbial carboxylesterases. The three-dimensional structure was modeled by sequence alignment and compared with others carboxylesterases. The topological differences suggested the classification of this enzyme and other Geobacillus-related carboxylesterases in a new {alpha}/β hydrolase family different from IV and VI.

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology-A, Faculty of Biology, University of Murcia, Campus Espinardo, E30011 Murcia, Spain. Phone: 34968364777. Fax: 34968364147. E-mail: alvaro{at}um.es

{triangledown} Published ahead of print on 20 March 2009.

Journal of Bacteriology, May 2009, p. 3076-3085, Vol. 191, No. 9
0021-9193/09/$08.00+0     doi:10.1128/JB.01060-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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