Identification of a hot-spot to enhance Candida rugosa lipase thermostability by rational design methods

Lipase is one of the most widely used classes of enzymes in biotechnological applications and organic chemistry. Candida rugosa 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.