Zhao H, Arnold F H
Division of Chemistry and Chemical Engineering 210-41, California Institute of Technology, Pasadena 91125, USA.
Protein Eng. 1999 Jan;12(1):47-53. doi: 10.1093/protein/12.1.47.
We used directed evolution to convert Bacillus subtilis subtilisin E into an enzyme functionally equivalent to its thermophilic homolog thermitase from Thermoactinomyces vulgaris. Five generations of random mutagenesis, recombination and screening created subtilisin E 5-3H5, whose half-life at 83 degrees C (3.5 min) and temperature optimum for activity (Topt, 76 degrees C) are identical with those of thermitase. The Topt of the evolved enzyme is 17 degrees C higher and its half-life at 65 degrees C is >200 times that of wild-type subtilisin E. In addition, 5-3H5 is more active towards the hydrolysis of succinyl-Ala-Ala-Pro-Phe-p-nitroanilide than wild-type at all temperatures from 10 to 90 degrees C. Thermitase differs from subtilisin E at 157 amino acid positions. However, only eight amino acid substitutions were sufficient to convert subtilisin E into an enzyme equally thermostable. The eight substitutions, which include known stabilizing mutations (N218S, N76D) and also several not previously reported, are distributed over the surface of the enzyme. Only two (N218S, N181D) are found in thermitase. Directed evolution provides a powerful tool to unveil mechanisms of thermal adaptation and is an effective and efficient approach to increasing thermostability without compromising enzyme activity.
我们利用定向进化将枯草芽孢杆菌枯草杆菌蛋白酶E转化为一种功能上等同于其嗜热同源物——来自普通嗜热放线菌的嗜热酶的酶。经过五代随机诱变、重组和筛选，产生了枯草杆菌蛋白酶E 5-3H5，其在83℃下的半衰期（3.5分钟）和活性最适温度（Topt，76℃）与嗜热酶相同。进化后酶的Topt高17℃，其在65℃下的半衰期是野生型枯草杆菌蛋白酶E的200多倍。此外，在10至90℃的所有温度下，5-3H5对琥珀酰-Ala-Ala-Pro-Phe-对硝基苯胺的水解活性均高于野生型。嗜热酶与枯草杆菌蛋白酶E在157个氨基酸位置上存在差异。然而，仅八个氨基酸替换就足以将枯草杆菌蛋白酶E转化为一种同样耐热的酶。这八个替换包括已知的稳定突变（N218S、N76D）以及几个以前未报道过的突变，分布在酶的表面。在嗜热酶中仅发现两个（N218S、N181D）。定向进化为揭示热适应机制提供了一个强大的工具，并且是一种在不影响酶活性的情况下提高耐热性的有效且高效的方法。
