Departamento de Ciência e Tecnologia de Alimentos, Universidade Federal de Santa Catarina, Rod. Admar Gonzaga, 1346, 88034-001, Florianópolis, Brazil.
Mol Biotechnol. 2010 Oct;46(2):168-75. doi: 10.1007/s12033-010-9282-5.
Staphylococcus xylosus AF208229 lipase was expressed in E. coli containing an histidine-tag (WT-Val). In the present work, in order to check the importance of the residue 309 in the specific activity, the amino acid side chain residue valine 309 was substituted by aspartate or lysine through site-directed mutagenesis. Both mutant lipases (MUT-Lys and MUT-Asp) were expressed in E. coli and the recombinant histidine-tagged lipases were purified by immobilized metal ion affinity chromatography. The enzyme activity was determined using p-nitrophenyl butyrate as substrate and secondary structure content was evaluated by circular dichroism. MUT-Lys and MUT-Asp presented significant increase of lipase activity (P < 0.05) in comparison to WT-Val, although highest activities for the three enzymes were observed at the same pH and temperature (pH 9.0 and 42 degrees C). The wild type and mutant lipases presented high thermal stability, after 30 min of incubation at 80 degrees C all enzymes retained their initial activities.
表皮葡萄球菌 AF208229 脂肪酶在含有组氨酸标签（WT-Val）的大肠杆菌中表达。在本工作中，为了检查残基 309 在比活性中的重要性，通过定点突变将氨基酸侧链残基缬氨酸 309 替换为天冬氨酸或赖氨酸。两种突变脂肪酶（MUT-Lys 和 MUT-Asp）在大肠杆菌中表达，并通过固定化金属离子亲和层析纯化重组组氨酸标记脂肪酶。使用对硝基苯丁酸作为底物测定酶活性，并通过圆二色性评估二级结构含量。与 WT-Val 相比，MUT-Lys 和 MUT-Asp 的脂肪酶活性显著增加（P < 0.05），尽管三种酶的最高活性在相同的 pH 和温度（pH 9.0 和 42°C）下观察到。野生型和突变型脂肪酶表现出较高的热稳定性，在 80°C 孵育 30 分钟后，所有酶都保留了其初始活性。
