Yang Deng-Feng, Wei Yu-Tuo, Huang Ri-Bo
Guangxi Academy of Sciences, Nanning, China.
Biosci Biotechnol Biochem. 2007 Mar;71(3):746-53. doi: 10.1271/bbb.60576. Epub 2007 Mar 7.
Using computer-aided design of single-site mutations, three amino acid residues determined by changes in folding free energy between wild-type (wt) and mutant proteins were exchanged to enhance the stability of pyruvate formate-lyase (PFL). The mutant enzymes were tested for properties such as optimum temperature, optimum pH, kinetic parameters, and stability to temperature. There were two mutant variants, Glu336Cys and Glu400Ile, that exhibited increased thermostability as compared to the wt enzyme. The melting temperatures (T(m), the temperature at which 50% inactivation occurs after heat treatment for 20 min) of Glu336Cys and Glu400Ile increased by 3.7 and 2.2 respectively. They also showed an increase in half life of about 1.80 and 2.21-fold, whereas Ala273Cys showed a slight decrease as compared with the wt enzyme.
利用单点突变的计算机辅助设计，交换了野生型（wt）和突变型蛋白质之间折叠自由能变化所确定的三个氨基酸残基，以提高丙酮酸甲酸裂解酶（PFL）的稳定性。对突变酶进行了最适温度、最适pH、动力学参数和热稳定性等性质的测试。有两个突变变体，即Glu336Cys和Glu400Ile，与野生型酶相比，它们表现出更高的热稳定性。Glu336Cys和Glu400Ile的解链温度（T(m)，热处理20分钟后50%失活时的温度）分别升高了3.7和2.2。它们的半衰期也分别增加了约1.80倍和2.21倍，而Ala273Cys与野生型酶相比略有下降。
