【摘要】 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%.更多还原

【Abstract】 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 7.5×104 by SDS-PAGE analysis,while the mass is 6.0×104 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.更多还原