Effects Of The Novel Disulfide Bonds On Structure And Functions Of Aspergillus Niger Phytase

The supplementation of animal feed with phytase can increase the bioavailability of phosphorus in monogastric animals besides reducing the phosphorus pollution. Improving the stability of phytase is of great urgency. As the development of gene engineering, it is to be a promising approach to improve enzyme property via protein engineering. The stable structural conformation is the base of biological function of proteins. Disulfide bonds, covalent linkage in protein structure, are important in the conformational stability of proteins. There are five pairs of disulfide bonds in phytase: Cys12-Cys21, Cys52-Cys395, Cys196-Cys446, Cys245-Cys263, Cys417-Cys425.By the software and site-mutation, the novel disulfide bonds were introduced into phytase, via the research of the effects of the novel disulfide bonds on the structure and functions of phytase, we discusse the problems of the introduction of the new disulfide bonds, set up a available strategy and offer a theory for the protein design of the enzymes.1. Using protein blast with A.niger (ficuum) phytase, we got the 3D model of the phytase. Software Disulfide by Design^TM was used to design mutation positions according to a model protein which has a 97% homology in amino acids with our phytase. According Swiss-PdbViewer and Ramachandran plot, the following positions, Tyr19-Pro34, Tyr 32-Ser246, Pro111-Ser253 and Pro193-Ser318, were most likely to form disulfide bonds if the relative amino acids were mutated to cysteines.Six mutant phytases were constructed and expressed in Pichia. Pastoris successfully.2. The phytases were purified and compared with WT phytase, the mutant phytases had no obvious change though SDS-PAGE analysis. The treatment of DTNB showed that there were no free cysteines in the mutant phytase and the novel disulfide bonds were formed successfully. As the activity of the mutant phytases was not changed obviously after the denaturation by DTT and the renaturation by DsbA, the native disulfide bonds were formed correctly.3. Compared the specific activity of mutant phytases with WT, the mutant MP111/253 was similar to the wild-type phytase, and the mutant MP193/318 decreased about 20%. The kinetics analysis showed that the K_m of the mutant phytases decreased obviously, which demonstrated that the mutant could enhance the affinity of phytase to phytate.4. The optimum pH study of phytases showed that there were two peak values (pH2.5 and pH5.0) in the curve of MP111/253 and two peak values (pH2.0 and pH5.0) in the curve of MP193/318. At pH5.0 to pH6.0, the MP111/253 retained more than 95% of its activity, whereas the wild-type phytase retained 75% at pH6.0. These results confirmed that there is wider pH in the mutant phytases than the wild-type phytase.5. The thermalstability compare of the WT and mutant phytases: At 60°C, the MP193/318 retained 72% of its activity, whereas the wild-type phytase retained 60% and the MP111/253 retained 50%. As the temperature increasing, the activity was decreased, after the treatment at 80°C for 15min, the wt and mutant phytases remained about 40%.