Studies On The Role Of Disulfide Bonds Of Phytase By Site-directed Mutagenesis

The supplementation of animal feed with phytase can increase the bioavailability of phosphorus in monogastric animals besides reducing the phosphorus pollution. Increasing the expression and improving the stability of phytase are of great urgency. As the development of gene engineering, it is to be a promising approach to improve enzyme property via protein engineering. Disulfide bonds are indispensable for the stability and function of enzyme. Phytase contains five pairs of disulfide bonds:Cys12-Cys21, Cys52-Cys395, Cys196-Cys446, Cys245-Cys263, Cys417-Cys425. To provide theoretical basis for improving stability by introducing disulfide bond, we studied the contribution of disulfide bonds to the catalytic activity, conformational stability and thermostability of phytase.The previous study indicated that the disulfide bonds played an important role in the catalytic activity and conformational stability of phytase. By three single mutations, three pairs of disulfide bonds were deleted, and two pairs of disulfide bonds were consided to be critical for the thermostability of phytase. To confirm the role of all the five pairs of disulfide bonds, we got six other mutant phytase (four single mutations and two double mutations) in this study. The enzymological characteristics of the wild-type phytase and nine mutant phytase were studied, and the main results were as follows:1. The kinetics analysis showed that the deletion of either Cys12-Cys21 or Cys196-Cys446 decreased the catalytic activity and affinity to subatrate, while the optimal pH were also changed. The deletion of Cys12-Cys21, Cys52-Cys395 and Cys196-Cys446 made the optimal temperature to be changed from 50℃to 45℃,55℃,40℃, resepectively.2. Conformation studies indicated that the deletions of the five disulfide bonds changed the structure of phytase more or less, espesically the deletion of Cys52-Cys395.3. Thermostability analysis indicated that the deletion of Cys52-Cys395, Cys245-Cys263 and Cys417-Cys425 decreased the thermolstability largely. The deletion of Cys196-Cys446 resulted an increase of the thermostability.