Li Guanlin, Chen Yuan, Fang Xingrong, Su Feng, Xu Li, Yan Yunjun
Key Laboratory of Molecular Biophysics, The Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology Wuhan 430074 P. R. China
RSC Adv. 2018 Jan 9;8(4):1948-1957. doi: 10.1039/c7ra11679a. eCollection 2018 Jan 5.
Lipase is one of the most widely used classes of enzymes in biotechnological applications and organic chemistry. lipases (CRL) can catalyze hydrolysis, esterification and transesterification with high regio-, stereo- and enantio-selectivity. However, thermal inactivation above 45 °C limits CRL's applications. Studies on improving the thermal stability of CRL are often limited by its slow-growing eukaryotic expression host, which is not suitable for large-scale screening. Identification of thermally stable mutants by rational design, regarded as an efficient substitution of experimental efforts, would provide a method for site-directed improvement of CRL. In this study, mutation-induced stability changes in CRL Lip1 were predicted by three rational design methods. Followed by conservative analyses and functional region exclusion, five mutants of a hot-spot, Asp457Phe, Asp457Trp, Asp457Met, Asp457Leu, and Asp457Tyr, were identified and prepared for enzymatic characterization. These five mutants increased the apparent melting temperature of Lip1 from 7.4 °C to 9.3 °C, with the most thermostable mutant, Asp457Phe, exhibiting a 5.5-fold longer half-life at 50 °C and a 10 °C increase in optimum temperature. Furthermore, pH stability of Lip1 was also enhanced due to the introduction of Asp457Phe mutation. The study demonstrates that thermally stable mutants of CRL could be identified with limited experimental efforts using rational design methods.
脂肪酶是生物技术应用和有机化学中使用最广泛的酶类之一。脂肪酶（CRL）能够以高区域选择性、立体选择性和对映体选择性催化水解、酯化和转酯反应。然而，45℃以上的热失活限制了CRL的应用。对提高CRL热稳定性的研究常常受到其生长缓慢的真核表达宿主的限制，这种宿主不适合大规模筛选。通过合理设计鉴定热稳定突变体，被认为是对实验工作的有效替代，将为CRL的定点改进提供一种方法。在本研究中，通过三种合理设计方法预测了CRL Lip1中突变诱导的稳定性变化。随后进行保守性分析和功能区排除，鉴定出一个热点的五个突变体，即Asp457Phe、Asp457Trp、Asp457Met、Asp457Leu和Asp457Tyr，并对其进行酶学特性分析。这五个突变体将Lip1的表观解链温度从7.4℃提高到9.3℃，其中最耐热的突变体Asp457Phe在50℃下的半衰期延长了5.5倍，最适温度提高了10℃。此外，由于引入了Asp457Phe突变，Lip1的pH稳定性也得到了增强。该研究表明，使用合理设计方法可以通过有限的实验工作鉴定出CRL的热稳定突变体。
