开盖型扩展青霉脂肪酶的分子设计及其界面激活分子机制的研究

项目名称： 开盖型扩展青霉脂肪酶的分子设计及其界面激活分子机制的研究

项目编号： No.30870545

项目类型： 面上项目

立项/批准年度： 2009

项目学科： 金属学与金属工艺

项目作者： 舒正玉

作者单位： 福建师范大学

项目金额： 31万元

中文摘要： 通过比较脂肪酶盖子结构及其两侧铰链区的氨基酸序列与对应酯酶区域的氨基酸序列，根据酯酶的氨基酸序列并结合氨基酸性质，运用基因工程手段，分别构建出pel-T66G, pel-T81P, pel-K94E, pel-Eα anl-S84G, anl-D99P, anl-K108E和anl- Eα#31561;八个脂肪酶基因突变体序列。上述突变脂肪酶基因序列导入Pichia pastoris GS115，经诱导表达并纯化后，进行酶学性质检测分析。 活性检测结果表明：PEL-Eα#21644;ANL-Eα#23436;全失活；PEL-T66G, PEL-T81P, PEL-K94E, ANL-S84G和ANL-K108E活性明显下降；而ANL-D99P水解pNPP的活性提高了2.2倍。 热稳定性检测结果表明：PEL-T66G，PEL-T81P，PEL-K94E和ANL-K108E的热稳定性没有显著变化；ANL-S84G和ANL-D99P的热稳定性显著降低； pNPB浓度对脂肪酶及其突变体水解活性的影响检测结果表明：ANL-D99P和ANL-K108E水解活性不再依赖油水界面激活。

中文关键词： 脂肪酶；酯酶；盖子结构；界面激活；分子设计

英文摘要： The amino acid sequence of the lid domain and the hinge region at both sides of the lid domain from lipase was compared with that of esterase, and then the difference of the amino acid sequence and the chatacteristic of the amino acid between lipase and esterase were identified. Based on these information, eight mutant lipase gene were constructed, including pel-T66G, pel-T81P, pel-K94E, pel-Eα anl-S84G, anl-D99P, anl-K108E and anl-Eα All of these mutant lipase genes were transformated into Pichia pastoris GS115 and the corresponding P. pastoris transformants were selected and induced by methanol. The recombinant proteins were purified to homogeneity and the activity and the stability of the recombinant protein was determined, respectively. The results of the activity analysis were shown as follows: the activity of PEL-Eαand ANL-Eαwas completedly lost, the activity of PEL-T66G, PEL-T81P, PEL-K94E, ANL-S84G and ANL-K108E was obviously decreased, while the hydrolysis activity of ANL-D99P towards pNPP increased 2.2-fold; The results of the temperature stability analysis were shown as follows: the temperature stability of PEL-T66G，PEL-T81P，PEL-K94E and ANL-K108E were similar to those of native lipase while the temperature stability of ANL-S84G and ANL-D99P were obviously decreased; The effects of pNPB concentration on the specific hydrolytic activity of lipases and lipase mutants were determined and the results were shown as follows: the hydrolytic activity of ANL-D99P and ANL-K108E were independent on the pNPB concentration and ANL-D99P and ANL-K108E displayed no interfacial activation.

英文关键词： Lipase; Esterase; Lid; Interfacial activation; Molecular design