关键词： 枯草芽孢杆菌, 植酸酶, phyC基因, pPIC3. K 表达载体, 毕赤巴斯德酵母, 胞内表达

Abstract: The Bacillus phytase is very suitable to be used in animal feed particularly in carp feed, because its optimum pH is at the neutral range and it has the excellent thermostability.At present, all the expressions of Bacillus phytase were reported in prokaryotic expression systems such as Bacillus and Escherichia coli, but the yield was very limited. So, the eukaryotic P. pastoris may be another better expression system due to its highly efficient expression for heterogenous protein.As the foundation of secretory expression in P. pastoris, the intracellular expression of Bacillus phytase was reported firstly in P .
pastoris . In this study, the phyC encoding phytase from Bacillus subtilisWHNB02 without signal peptide sequencewas ampl-ified by polymerase chain reaction (PCR) .The phyC fragment was sequenced and cloned into pPIC3. 5K expression vector of P. pastoris .The pPIC3. 5KphyC plasmid was linearized and transformed into Pichia Pastoris GS115 strain by electroporation.Positive strain was screened and purified by culturing on MD plate andMM plate. Furthermore, it was cultured and induced by 0. 5% methanol. SDS- PAGE analysis shows that phytase is expressed intracellularly, and the yield comes to 24% of the total soluble protein and its molecular weight is 42. 01 KD.The enzymatic analysis shows that the expression product has the biological activity of native phytase, its opt imum reaction pH and temperature is 7. 5 and 70 ?? , respectively.Moreover, its residual activity with treatment at 90 ?? for 10min is 42%.All of these properties are better than the native phytase from Bacillus subtilis.

Key words: bacillus subtilis, phytase, phyC, pPIC3. 5K expression vector, Pichia pastoris, intracellular expression