Purification And Characterization Of Two Phytases Expressed By Pichia Pastoris And Study Of Their N-Glycosylation

Two types of ecto-phytase, which were isolated and characterized from recombinant Pichia pastoris yeast, were studied on their N-glycosylation, crystal birth and renaturation. The two phytases, CMⅠand CMⅡ, were isolated from the fermentation of phytase by gene recombinant with one step of CM-Sephadex C-50 chromatography. They were identified to be homogeneous with SDS-PAGE and the partial N-terminal amino acid sequences.The molecular weight of CMⅠand CMⅡ were 55.3kD and 53.1kD respectively, and their optimum temperature and pH were the same as 55℃ and 5.0. The Km values of CMⅠunder 37℃ was 0.42mmol/L, and the Km values of CMⅡunder 37℃ was 0.79mmol/L. The activity of CMⅠand CMⅡ could be inhibited by Cu2+, Fe2+, Al3+, and Zn2+ . The specific activity of CMⅡ were higher than that of CMⅠ. The partial N-terminal amino acid sequence of two phytases were identical .The N-glycosylation analysis was performed with Endoglycosidase H, CMⅠ and CMⅡwere about 13.1% and 10.9% glycosylated respectively (calculations were made on the basis of original molecular weight before deglycosylation). It was testified that the difference between their molecular weight were induced by the different extents of N-glycosylation. After deglycosylation, it was found that the specific activity of CMⅠwas reduced to 81 percent of original specific activity of CMⅠ, while deglycosylation had little effect on the specific activity of CMⅡ. And the specific activity of CMⅡ was still higher than that of CMⅠ after deglycosylation. It showed that N-glycosylation of the phytases could stabilitate, even enhance the specific activity of the two phytases. According to the Crystal Screen, crystal birth was tested with 38 kinds of crystal conditions. Under the 12nd condition (i.e. 30% 2-Propanol, 0.1mol/L NaHepes pH7.5, and 0.2 mol/L MgCl2), the crystal of CMⅡ was obtained.Bae et al reported a staining method for detecting phytase activity. Here the method was improved, and a novel SDS-PAGE counterstaining, which was SDS-PAGE and ferrous sulfate-ammonium molybdate counterstaining, was established to detect phytase activity specially.After thermal denaturation and SDS-PAGE, the phytases could recover their activity partially. The mechanism of renaturation was studied with SDS-PAGE counterstaining. It showed that Triton X-100 and low temperature had played key roles in refolding the phytases. This provided a new evidence for understanding protein refolding mechanism and the theory of the primary structure of proteins determining the advanced structure.