酸性植酸酶phyB在毕赤酵母GS115中的表达

Studies on Expression of Acid Phosphorutase phyB Gene in Pichia pastoris GS115

通过PCR方法扩增得到无信号肽无内含子的酸性植酸酶phyB基因,将此基因克隆至毕赤酵母表达载体pPIC9K上,并转化毕赤酵母GS115感受态细胞,获得重组毕赤酵母.在甲醇的诱导下,该植酸酶phyB基因在毕赤酵母中得到表达.经SDS-PAGE凝胶电泳分析,该重组蛋白的相对分子质量为7.5×104,而该植酸酶在大肠杆菌DH5α表达系统中表达的相对分子质量为6.0×104,这可能是由于糖基化的结果.酶学性质检测发现,表达的植酸酶最适作用温度为65℃,比野生原始菌株的提高了10℃;诱导pH值为5.5时,蛋白的表达量最高,为10 528.6 U/mL,是野生菌株的2.8倍;最适作用pH值为2.5;在70℃条件下处理60 min,该酶的相对酶活力为40%,在75℃时处理10 min,该酶的相对酶活力为10%.

The phyB gene without signal peptides or introns has been amplified by PCR using the cDNA as templates, then inserted into pPIC9k to obtain the recombinant vector pPIC9k-phyB. The recombinant vectors were transformed into competent cell P. pastoris GS115. Induced by methanol, the phyB gene was expressed and the molecular mass of the muture phyB was determined to be 7.5×10^4 by SDS-PAGE analysis, while the mass is 6.0×10^4 as phyB gene expressed in E.coli, which may be caused by the glycosylation of the yeast cell. The analysis of enzymatic peoperties shows that its optimal temperature is 65 ℃, 10 ℃ higher than phyB produced from wide type strain; and its optimal induce pH and optimal reaction pH are 5.5 and 2.5 respectively; the expression level of phyB can reach 10 528.6 U/mL, 2.8-fold more than wide type strain. Under the condition of 70 ℃ for 60 min, the phyB remains 40% of the initial activity, it can retain only 10% of its activity after denaturation at 75 ℃ for 10 min.