To improve the proteolytic stability of the lipase LIP2 from Yarrowia lipolytica, the peptide bonds susceptible to trypsin in LIP2 were analyzed by tandem mass spectrometry and redesigned by site-directed mutagenesis. Different variants of the enzyme were expressed in Pichia pastoris GS115 and their biochemical properties were subsequently investigated. Although most of the variants were still cleaved by trypsin, some of them did show an evident increase of resistance against proteolytic degradation. The most stable mutant was LIP2-C5, in which five trypsin-cleavage sites were replaced by non-preferred amino acids. Upon incubation with human trypsin for 80 min at 37°C, the mutant LIP2-C5 was found to retain >70% of its initial activity, compared to only 10% for the wild-type.

中文翻译：

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解脂耶氏酵母脂肪酶的工程化变体，具有改进的胰蛋白酶抵抗力，可用于酶替代治疗。

为了提高解脂耶氏酵母（Yarrowia lipolytica）的脂肪酶LIP2的蛋白水解稳定性，通过串联质谱分析了LIP2中对胰蛋白酶敏感的肽键，并通过定点诱变进行了重新设计。该酶的不同变体在巴斯德毕赤酵母GS115中表达，随后对其生化特性进行了研究。尽管大多数变体仍被胰蛋白酶切割，但其中一些确实显示出对蛋白水解降解的抗性明显增加。最稳定的突变体是LIP2-C5，其中五个胰蛋白酶切割位点被非优选氨基酸取代。与人胰蛋白酶在37°C孵育80分钟后，发现突变体LIP2-C5保留了其初始活性的70％以上，而野生型仅为10％。