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| Content Provider | PubMed Central |
|---|---|
| Author | Silvia, Montoro-garcía Irene, Martínez-martínez José, Navarro-fernández Takami, Hideto Francisco, García-carmona Sánchez-ferrer, Álvaro |
| Abstract | 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 α/β hydrolase family different from IV and VI. |
| Related Links | http://dx.doi.org/10.1128/JB.01060-08 |
| Starting Page | 3076 |
| File Format | |
| ISSN | 10985530 |
| e-ISSN | 10985530 |
| Journal | Journal of Bacteriology |
| Issue Number | 9 |
| Volume Number | 191 |
| Language | English |
| Publisher | American Society for Microbiology (ASM) |
| Publisher Date | 2009-05-01 |
| Access Restriction | Open |
| Rights Holder | American Society for Microbiology (ASM) |
| Subject Keyword | Research in Higher Education |
| Content Type | Text |
| Resource Type | Article |
| Subject | Molecular Biology Microbiology |
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