微生物学杂志
微生物學雜誌
미생물학잡지
Journal of Microbiology
2015年
4期
32-36
,共5页
钟开新%陈丽芝%李阳源
鐘開新%陳麗芝%李暘源
종개신%진려지%리양원
α-半乳糖苷酶%里氏木霉%毕氏酵母%高密度发酵
α-半乳糖苷酶%裏氏木黴%畢氏酵母%高密度髮酵
α-반유당감매%리씨목매%필씨효모%고밀도발효
α-galactosidase%Trichoderma reesei%Pichia pastoris%high density fermentation
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。
通過反轉錄PCR從裏氏木黴RutC－30基因中剋隆到α－半乳糖苷酶基因agl2，將其構建到具有AOX1彊啟動子的錶達載體pPIC9K中，線性化後轉化到畢氏酵母GS115中進行錶達。結果錶明agl2基因在畢氏酵母GS115中得到瞭高效錶達，在50 L髮酵罐中高密度髮酵168 h，酶活達最高值1106 U／mL。經PCR產物鑒定，結果錶明agl2基因已整閤到畢氏酵母GS115基因組中。
통과반전록PCR종리씨목매RutC－30기인중극륭도α－반유당감매기인agl2，장기구건도구유AOX1강계동자적표체재체pPIC9K중，선성화후전화도필씨효모GS115중진행표체。결과표명agl2기인재필씨효모GS115중득도료고효표체，재50 L발효관중고밀도발효168 h，매활체최고치1106 U／mL。경PCR산물감정，결과표명agl2기인이정합도필씨효모GS115기인조중。
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.
