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
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
复制标题
DOI：
10.1016/j.bbapap.2003.09.008
复制
发表时间：
2004-01-14
影响因子：
3.2
通讯作者：
Esaki, N
中科院分区：
文献类型：
--
作者：
Kulakova, L;Galkin, A;Esaki, N
对分离自南极土壤的嗜冷菌Psychrobacter sp. Ant 300的酯酶基因进行了克隆、测序并在大肠杆菌中表达。PsyEst是脂肪酶/酯酶家族中对脂肪酶敏感的脂肪酶（HSL）组的成员，是一种冷活性、热不稳定的酶，具有在低温（5-25 ℃）下的高催化活性、低活化能（例如，4.6 kcal/ mol，用于对硝基苯基丁酸酯的水解），在40 ℃和pH 7.9下孵育期间热灭活的t（1/2）值为16 min。PsyEst的三维结构模型预测Gly（244）位于PsyEst活性位点附近的环中，并且该氨基酸残基被脯氨酸取代可能会使酶的活性位点环境刚性化。因此，我们将Gly（244）-> Pro取代引入酶中。稳定性研究表明，突变体在40 ℃和pH 7.9孵育期间的热失活的t（1/2）值为11.6 h，这显著大于野生型酶。突变体的k（cat）/K-m值低于野生型的值。此外，这种氨基酸取代引起的转移的酰基链长度特异性的酶向更高的偏好短链脂肪酸酯。所有这些观察结果可以解释的突变所带来的活性位点的灵活性降低，并与冷活性和热不稳定性产生的酶的活性位点周围的本地灵活性的假设是一致的。(C)2003 Elsevier B. V.保留所有权利。
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 degreesC), 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 degreesC 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 degreesC 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. (C) 2003 Elsevier B.V. All rights reserved.