| Glycosylation is one of the ways for the post-translational modifications of Eukaryotic cells, which is extremely important for structure and function of proteins. The phytase from Aspergillus belongs to Histidine Acid Phosphatase family (EC.3.1.3.2), including a typical conserved motif of RHGARYP, which was related to catalytic activity of the enzyme. Previous results showed that the phytase from this family was a glycosylated protein with approximately 10 potential glycosylation sites.The activity of the phytase depends on the glycosylation modification,However the significance of this modification to the enzyme and the roles of different glycosylation sites in the catalytic reaction has not been reported.To study the glycosylation sites of the phytase from Aspergillus niger 963 in the significance of its enzymatic properties, we deleted two N-glycosylation sites of phyA2 gene by Megaprimer PCR-mediated gene site-directed mutagenesis techniques,and the mutants were expressed in the Pichia pastoris successfully. Main results follow as:(1) Using the cloned phyA2 gene from Aspergillus niger 963 as a template,we deleted two glycosylation sites by Megaprimer PCR-mediated gene site-directed mutagenesis techniques in the nucleic acid level. We also constructed expression vector pPIC9-N87Q, pPIC9-N102Q.The recombinant protein were expressed in Pichia pastoris GS115 by fermentation.The SDS-PAGE and Western-blotting analysis of recombiant proteins showed that the apparent molecular weight of the mutants were dropped about lOkD.(2) The study of the optimum temperature and the thermalstability revealed that the optimum temperature of wild type phytase phyA2 and its mutants N87Q and N102Q were 50℃,but the N87Q had a higher activity between 45℃and 50℃compared with the WT.The N87Q retained more than 50% of its initial activity after incubation at 60℃for 1h, which was 20% lower than WT. N102Q retained more than 10% of its initial activity after 10 mins, but its activity was completely lost after 15 mins; after incubation at 70℃for 2 mins, both WT and N87Q retained more than 80% of its initial activity, but N102Q was only 25%. Afterward their activity was maintained at this level; Pre-treatment 2 mins at 80℃, the activity of the WT and mutants were decreased to a lower level, but increased by 10% after 4 mins, and then, maintained a stable level. N102Q was more stable with the increased temperature. This is a surprising phenomenon.(3) The study of the optimum pH and pH-stability, there were two optimum pH peaks for the WT and N87Q at pH2.0 and pH6.0, however the peaks of N102Q at pH2.5 and pH6.0, the activity of which at pH2.5 was lower than the peak of the WT at pH2.0. WT and N102Q retained more than 70% of its initial activity after being incubated under varying pH conditions; however the loss of activity for N102Q was more than 50% below pH 3.0 and 100% above pH 8.0.(4) The activity of wild-type phytase and its mutants could be increased by Fe2+,Fe3+,Ca2+,Co2+,Mn2+,K+和EDTA and inhibited by Mg2+,Zn2+,Cu2+,Ag+slightly inhibited the enzymatic reaction.but as a strong denaturant; SDS had a little effect on the activity.(5) There were no difference between wild-type phytase and its mutants in enzyme kinetic parameters, Specific activity was more than 100,000IU/mg, and Km=0.435mmol/l, Kmax=3,547,357.218IU/mg.min.
|
PDF Full Text Request