College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, People's Republic of China.
J Biotechnol. 2012 Dec 15;164(2):248-53. doi: 10.1016/j.jbiotec.2012.08.023. Epub 2012 Sep 7.
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.
解脂耶氏酵母脂肪酶 Lip2（YlLip2）是一种用途广泛的生物催化剂。然而，其实际应用常因稳定性低而受到限制。本研究采用三种互补的蛋白质工程策略来提高该酶的热稳定性。第一种策略是易错 PCR 定向进化，结果得到的 YlLip2 变体在 50°C 下的热失活动力学半衰期比野生型酶延长了 2.5 倍。第二种策略是使用所谓的 B 因子迭代测试（B-FIT）的半理性设计，发现了两种耐热性 YlLip2 变体，它们在 50°C 下的热失活动力学半衰期分别比野生型酶延长了 2 倍和 5 倍。第三种策略是使用定点突变，组合前两种策略中鉴定的所有三种耐热性突变，与野生型酶相比，YlLip2 的热失活动力学半衰期提高了 7 倍。这些工程化脂肪酶不仅为蛋白质结构和功能关系提供了新的见解，而且为实际应用提供了潜在有用的催化剂。
