Improving the thermostability of lipase Lip2 from Yarrowia lipolytica.

Yarrowia lipolytica lipase Lip2 (YlLip2) is a highly versatile biocatalyst. However, its practical use is often hampered by its low stability. Here three complementary protein engineering strategies were used to improve the thermostability of this enzyme. The first strategy was error-prone PCR based directed evolution, which resulted in a YlLip2 variant with a 2.5-fold longer half-life of thermal inactivation at 50°C compared to the wild-type enzyme. The second strategy was semi-rational design using the so-called B-factor iterative test (B-FIT), which led to the discovery of two thermostable YlLip2 variants that showed a half-life of thermal inactivation 2-fold and 5-fold longer than that of the wild-type enzyme, respectively, at 50°C. The third strategy was to use site-directed mutagenesis to combinatorially combine all three thermostabilizing mutations identified in the first two strategies, which improved the half-life of thermal inactivation of YlLip2 by 7-fold compared to that of the wild-type enzyme. Such engineered lipases provide not only new insights on the protein structure and function relationship but also potentially useful catalysts for practical applications.