里氏木霉α－半乳糖苷酶基因agl2在毕氏酵母中高效表达

摘要

α-galactosidase gene ( agl2 ) , amplified by reverse transcription PCR from genome of Trichoderma reesei was cloned into plasmid pPIC9K which had a strong promoter AOX1 and transferred and expressed in Pichia pastoris GS115 after linearization.The results showed that agl2 gene was highly expressed in P.pastoris.Theα-galactosidase activity reached at the highest value of 1 106 U/mL methanol induction in 50 L high-density fermentation for 168 h. The agl2 gene had integrated into the genome of P.pastoris GS115 proved by PCR.%通过反转录PCR从里氏木霉RutC－30基因中克隆到α－半乳糖苷酶基因agl2，将其构建到具有AOX1强启动子的表达载体pPIC9K中，线性化后转化到毕氏酵母GS115中进行表达。结果表明agl2基因在毕氏酵母GS115中得到了高效表达，在50 L发酵罐中高密度发酵168 h，酶活达最高值1106 U／mL。经PCR产物鉴定，结果表明agl2基因已整合到毕氏酵母GS115基因组中。