芽孢杆菌植酸酶基因在毕赤酵母中的胞内表达

根据已知的枯草芽孢杆菌WHNB02植酸酶phyC基因全序列设计一对引物,采用PCR法从含有该基因的pUC18-phyC质粒上获得了长约1.1kb不含有信号肽序列的植酸酶phyC基因表达片段.经T载体克隆及序列测定后,构建毕赤巴斯德表达载体pPIC3.5K-phyC,并电转化毕赤巴斯德酵母宿主菌GS115.经MD和MM平板筛选、酶活性测定,获得了阳性转化子,并进行了诱导表达.SDS-PAGE分析表明:表达产物分子量为42.01KD,表达量占细胞可溶性总蛋白的24%,并具有植酸酶的生物学活性.酶学性质分析结果显示:胞内表达的植酸酶酶促反应最适pH值为7.5;最适反应温度为70℃;经90℃处理10min,残留酶活性42% 均优于出发菌株天然植酸酶的相应性质.

Intracellular Expression of Bacillus Subtilis Phytase phyC in Pichia Pastoris

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 subtilis WHNB02 without signal peptide sequence was amplified 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 and MM 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 optimum reaction pH and temperature is 7.5 and 70 ℃,respectively.Moreover,its residual activity with treatment at 90 ℃ for 10 min is 42%.All of these properties are better than the native phytase from Bacillus subtilis.