Abstract：
SshEstI, a carboxylesterase from the thermoacidophilic archaeon Saccharolobus shibatae, is a member of the hormone-sensitive lipase family that displays slightly alkaliphilic activity with an optimum activity at pH 8.0. In this study, three distinct strategies were explored to confer acidophilic properties to SshEstI. The first strategy involved engineering the oxyanion hole by replacing Gly81 with serine or aspartic acid. The G81S mutant showed optimum activity at pH 7.0, whereas the aspartic acid mutant (G81D) rendered the enzyme slightly acidophilic with optimum activity observed at pH 6.0; however, kcat and kcat/Km values were reduced by these substitutions. The second strategy involved examining the effects of surfactant additives on the pH-activity profiles of SshEstI. The results showed that cetyltrimethylammonium bromide (CTAB) enhanced wild-type enzyme (WT) activity at acidic pH values. In the presence of 0.1 mM CTAB, G81S and G81D were acidophilic enzymes with optimum activity at pH 6.0 and 4.0, respectively, although their enzyme activities were low. The third strategy involved engineering the active site to resemble that of kumamolisin-As (kuma-As), an acidophilic peptidase of the sedolisin family. The catalytic triad of kuma-As was exchanged into SshEstI using site-directed mutagenesis. X-ray crystallographic analysis of the mutants (H274D and H274E) revealed that the potential hydrogen donor-acceptor distances around the active site of WT were fully maintained in these mutants. However, these mutants were inactive at pH 4-8.
摘要：
SshEsti,一种来自嗜酸热古细菌的羧酸酯酶，是激素敏感性脂肪酶家族的成员，在pH8.0时表现出轻微的嗜碱性活性。在这项研究中,探索了三种不同的策略来赋予SshEstI嗜酸特性。第一种策略涉及通过用丝氨酸或天冬氨酸代替Gly81来改造氧阴离子孔。G81S突变体在pH7.0时显示出最佳活性，而天冬氨酸突变体（G81D）使该酶略微嗜酸性，在pH6.0时观察到最佳活性；但是，这些替换降低了kcat和kcat/Km值。第二种策略涉及检查表面活性剂添加剂对SshEstI的pH-活性曲线的影响。结果表明，十六烷基三甲基溴化铵（CTAB）在酸性pH值下增强了野生型酶（WT）的活性。在0.1mMCTAB的存在下，G81S和G81D是嗜酸性酶，分别在pH6.0和4.0时具有最佳活性，尽管它们的酶活性很低。第三种策略涉及将活性位点设计为类似于kumamolisin-As（kuma-As），sedolisin家族的一种嗜酸肽酶。使用定点诱变将kuma-As的催化三联体交换为SshEstI。突变体（H274D和H274E）的X射线晶体学分析显示，在这些突变体中，WT活性位点周围的潜在氢供体-受体距离得到了完全维持。然而,这些突变体在pH4-8时无活性。
