Cysteine Engineering of an Endo-polygalacturonase from <i>Talaromyces leycettanus</i> JCM 12802 to Improve Its Thermostability
Sheng Wang, Kun Meng, Xiaoyun Su, Nina Hakulinen, Yaru Wang, Jie Zhang, Huiying Luo, Bin Yao, Huoqing Huang, Tao Tu
Abstract
Thermostable enzymes have many advantages for industrial applications. Therefore, in this study, computer-aided design technology was used to improve the thermostability of a highly active endo-polygalacturonase from Talaromyces leycettanus JCM12802 at an optimal temperature of 70 °C. The melting temperature and specific activity of the obtained mutant T316C/G344C were increased by 10 °C and 36.5%, respectively, compared with the wild-type enzyme. The crystal structure of the T316C/G344C mutant showed no formation of a disulfide bond between the introduced cysteines, indicating a different mechanism than the conventional mechanism underlying improved enzyme thermostability. The cysteine substitutions directly formed a new alkyl hydrophobic interaction and caused conformational changes in the side chains of the adjacent residues Asn315 and Thr343, which in turn caused a local reconstruction of hydrogen bonds. This method greatly improved the thermostability of the enzyme without affecting its activity; thus, our findings are of great significance for both theoretical research and practical applications.