生命科学学院知识库

尖孢镰刀菌胰蛋白酶（Fusarium oxysporum trypsin, FOT, 3.4.21.4）是一种由尖孢镰刀菌产生的丝氨酸蛋白酶，广泛的应用于工业、食品、医药等领域。本研究通过基因工程技术手段将尖孢镰刀菌来源的胰蛋白酶表达于毕赤酵母系统中，在该真核表达系统中通过限制性内切酶分别在胰蛋白酶的N端引入氨基酸YVEF（TE）及YV（TS），将野生型的尖孢镰刀菌中产生胰蛋白酶FOT作为对照组，研究在毕赤酵母表达系统中N端残留氨基酸的不同对酶学性质的影响。研究结果表明：重组胰蛋白酶及野生型胰蛋白酶的最适温度和pH均相同，分别为40°C和8.0。在最适反应温度（40°C )下研究胰蛋白酶的热稳定性，结果表明N端含有氨基酸YVEF和YV的重组酶TE和TS的热稳定性比野生型酶FOT的热稳定性显著的增加。此外，研究了pH对胰蛋白酶的稳定性的影响，结果显示重组胰蛋白酶TE和TS对pH的耐受性要高于野生型酶FOT。酶动力学研究分别表明：重组酶TE和TS的催化效率（指特异性常数，kcat / Km）比野生型FOT分别提高了1.75倍和1.23倍。在模拟的3D结构中发现，氨基酸YVEF和YV的引入对于胰蛋白酶的整体结构并没有显著的改变，但是氨基酸的引入导致了底物结合口袋（D174，G198和G208）与催化三联体（His42，Asp102和Ser180）之间的距离变短，从而提高电子转移速率和催化效率。与此同时，与野生型FOT相比，重组酶TE和TS的分子内氢键和阳离子π-相互作用力显著增加。这些结果表明，合理的设计N-端残留的肽可能是改善胰蛋白酶特性的一种有效的方法。

Fungal Fusarium oxysporum trypsin (FOT) is a serine protease similar to mammal trypsin., which is widely used in various fields such as industry, food and medicine. FOT could be successfully expressed in Pichia pastoris. In this study, the recombinant TE (with peptide YVEF) and TS (with peptide YV) expressed in Pichia pastoris were compared to the wildtype FOT of the enzyme properties. The results showed that the FOT, TE and TS displayed the same optimum temperature (40°C) and pH 8.0. However, the recombinant TE and TS respectively with the peptide YVEF and YV showed significantly increased thermostability at 40°C and 50 °C. Moreover, the recombinant TE and TS also showed the pH tolerance under alkaline conditions. Compared with the wildtype FOT, the intramolecular Hydrogen bonds and the cation π-interactions of the recombinant TE and TS were significantly increased. The recombinant TE and TS has also a significantly increased the catalytic efficiency (referring to the specificity constant, kcat /Km) 1.75-fold and 1.23-fold to the wild FOT. In silico modeling analysis uncovered that introduction of the peptide YVEF and YV resulted in a shorter distance between substrate binding pocket (D174, G198 and G208) and catalytic triad (His42, Asp102 and Ser180) which will improve the electron transfer rate and catalytic efficiency. The results suggested that engineering of the N-terminus with residual peptides might be an effective approach for improve the properties of the trypsin.