| Phytase catalyzes the hydrolysis of phytate to myo-inositol and inorganic phosphate. The supplementation of animal feed with phytase increases the bioavailability of phosphorus in monogastric animals besides reducing the phosphorus pollution. Despite its corroborated effect, the low yield and stability remain a critical issue in the extensive application of phytase. Therefore, increasing the yield and improving the stability of phytase are of great urgency.Studies of enhancing the stability of phytase are mainly focused on adding protecting agent or the immobility. 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 disulfide bonds. However, comparatively few studies regard the contribution of disulfide bonds to the catalytic activity and conformational stability.In this paper, we selected an A. niger strain which produces extracellular phytase and cloned the phyA gene. This gene was transformed into Pichia pastoris and phytase with potential industrial application was overexpressed. We also investigated the effects of the five disulfide bonds on the conformation, thermostability, resistance to denaturant and catalytic activity of phytase for the first time. Comparison of the functions of different disulfide bonds was also discussed comprehensively. The main results were as follows:1. A phytase-producing strain, Aspergillus niger N-J, was isolated and identified. The phyA gene was cloned by PCR with the genomic DNA of A. niger N-J as template. The sequence analysis showed that the ORF comprises 1347 bp, encoding 448 amino acid residues of phytase mature protein. It has the conservative motif RHGXRXP and 11 potential N-glycosylation sites. It also has 10 Cys that form five pairs of disulfide bonds. We transformed phyA into Pichia pastoris and overexpressed phytase with potential industrial application.2. In this study, five expression plasmids of phytase mutants with deletion of the five disulfide bonds respectively (C12S, C263S, C395S, C417S, C446S) were constructed. All the five mutants were expressed at high level in Pichia pastoris.3. Assay of the enzymological characteristics showed that all the five mutants hold high activity in the range of pH 2.5 to 5.5, which are similar to the stomach and intestines of animal. The deletion of Cys12-Cys21 and Cys245-Cys263 shrank the favorable pH range, and also the deletion of Cys12-Cys21 decreased the optimum temperature. The kinetics analysis showed that the deletions of Cys52-Cys395, Cys196-Cys446, Cys245-Cys263 and Cys417-Cys425 increased the kcat and the kcat/ Km; and the deletion of Cys12-Cys21 decreased the the kcat and the kcat/ Km.4. Conformation studies indicated that deletions of the five disulfide bonds changed the stucture of phytase more or less. The structure of C263S and C417S become loose while the C446S becomes more compact than the wild-type.5. The changes of the resistance to guanidine hydrochloride (GdnHCl) were studied by spectroscopic and activity analysis. It turns out the deletions of disulfide bonds decreased the stability of phytase in GdnHCl solution. However the effect varies greatly with different mutants. The removal of Cys12-Cys21, Cys245-Cys263 and Cys417-Cys425 decreased the stability in GdnHCl solution dramatically, while the removal of Cys52-Cys395 and Cys196-Cys446 changed relatively slighter.6. Thermostability analysis indicated that the deletion of disulfide bond caused a easier-to-change conformation of the active-site. The deletion of Cys245-Cys263 and Cys417-Cys425 decreased the thermolstability largely and the mutant C446S is more stable than the wild-type phytase in high temperature.
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