Cold-active esterase from Psychrobacter sp. Ant300: gene cloning, characterization, and the effects of Gly→Pro substitution near the active site on its catalytic activity and stability
✍ Scribed by Ljudmila Kulakova; Andrey Galkin; Toru Nakayama; Tokuzo Nishino; Nobuyoshi Esaki
- Book ID
- 104003244
- Publisher
- Elsevier Science
- Year
- 2004
- Tongue
- English
- Weight
- 333 KB
- Volume
- 1696
- Category
- Article
- ISSN
- 1570-9639
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✦ Synopsis
The gene encoding an esterase (PsyEst) of Psychrobacter sp. Ant300, a psychrophilic bacterium isolated from Antarctic soil, was cloned, sequenced, and expressed in Escherichia coli. PsyEst, which is a member of hormone-sensitive lipase (HSL) group of the lipase/esterase family, is a cold-active, themolabile enzyme with high catalytic activity at low temperatures (5 -25 jC), low activation energy (e.g., 4.6 kcal/ mol for hydrolysis of p-nitrophenyl butyrate), and a t 1/2 value of 16 min for thermal inactivation during incubation at 40 jC and pH 7.9. A three-dimensional structural model of PsyEst predicted that Gly 244 was located in the loop near the active site of PsyEst and that substitution of this amino-acid residue by proline should potentially rigidify the active-site environment of the enzyme. Thus, we introduced the Gly 244 ! Pro substitution into the enzyme. Stability studies showed that the t 1/2 value for thermal inactivation of the mutant during incubation at 40 jC and pH 7.9 was 11.6 h, which was significantly greater than that of the wild-type enzyme. The k cat /K m value of the mutant was lower for all substrates examined than the value of the wild type. Moreover, this amino-acid substitution caused a shift of the acyl-chain length specificity of the enzyme toward higher preference for short-chain fatty acid esters. All of these observations could be explained in terms of a decrease in active-site flexibility brought about by the mutation and were consistent with the hypothesis that cold activity and thermolability arise from local flexibility around the active site of the enzyme.