不依赖油水界面激活的黑曲霉脂肪酶突变体的构建

Construction of Aspergillus niger lipase mutants with oil-water interface independence

为获得不依赖油水界面激活的黑曲霉脂肪酶(ANL)突变体,在生物信息学分析基础上,对黑曲霉脂肪酶盖子结构域两侧铰链区的氨基酸残基进行了置换突变,获得两个黑曲霉脂肪酶突变体(ANL-Ser84Gly和ANL-Asp99Pro)。对不同浓度对硝基苯丁酸酯的水解活性检测结果表明:ANL-Ser84Gly的催化活性仍依赖油水界面,而ANL-Asp99Pro的催化活性不再依赖油水界面。底物特异性检测结果表明:较ANL而言,ANL-Ser84Gly的比活力显著降低,其水解对硝基苯棕榈酸酯、对硝基苯豆蔻酸酯、对硝基苯月桂酸酯和对硝基苯癸酸酯的比活力分别降低了29.8%,53.1%,60.1%和77.1%;而ANL-Asp99Pro水解对硝基苯棕榈酸酯的比活力提高了2.2倍。铰链区的突变破坏了突变体蛋白质分子ANL-S84G与ANL-D99P的二级结构作用力,使突变体分子的二级结构域更趋不稳定,从而导致了突变体分子的热稳定性显著降低。不依赖油水界面激活的脂肪酶突变体的构建,将有利于深入了解脂肪酶界面激活的分子机制。

Based on previous bioinformational analysis results,two Aspergillus niger lipase（ANL） mutants,ANL-Ser84Gly and ANL-Asp99Pro were constructed to screen ANL mutants with oil-water interface independence.ANL-Ser84Gly still displayed a pronounced interfacial activation,while ANL-Asp99Pro displayed no interfacial activation.The specific activity of ANL-Ser84Gly towards p-nitrophenyl palmitate（-myristate,-laurate and-decanoate） decreased by 29.8%（53.1,60.1 and 77.1,respectively） than that of ANL,while the specific activity of ANL-Asp99Pro towards p-nitrophenyl palmitate increased by 2.2-fold.The mutation in the hinge region at both sides of the lid domain also destabilized various secondary structure factors of ANL-S84G and ANL-D99P,which resulted in a substantial decrease in thermostability.The achievement to construct oil-water interface-independent ANL mutants would help to further understand lipase interfacial activation mechanism.