奶牛β-1,4-半乳糖基转移酶基因在毕赤酵母GS115中的转化表达及发酵优化  

Optimization of expression and fermentation of β-1,4-galactosyltransferase gene from dairy cow in Pichia pastoris GS115

作　　者：姚晶[1,2] 吴正钧[1] 任婧[1] 

机构地区：[1]乳业生物技术国家重点实验室,光明乳业股份有限公司技术中心,上海200436 [2]上海海洋大学食品学院,上海201306

出　　处：《江苏农业学报》2012年第6期1367-1372,共6页Jiangsu Journal of Agricultural Sciences

基　　金：国家科技部973计划(2010CB735705);上海市科委课题(09DZ2251400)

摘　　要：采用电转化法将已构建好的表达载体pPIC9K-GT转化到毕赤酵母(Pichina pastoris)GS115中。通过对电转化条件进行优化,达到了理想的转化效率,得到了大量转化子,并通过表型筛选及PCR鉴定,筛选到阳性转化子。对重组的P.pastoris GS115-GT进行了诱导表达,用SDS-PAGE法检测到目的蛋白质条带,证明β-1,4-半乳糖基转移酶基因(GT)在P.pastoris GS115中能够表达;用苯酚红法测定了粗酶液的活性,其比酶活为16.40 U/ml。对工程菌的发酵条件进行了初步优化,在最佳工艺下的比酶活达到30.909 U/ml,比优化前提高了88.47%。Recombinant plasmid pPIC9K-GT was transformed into P.pastoris GS115 by electroporation.In order to achieve a high transformation efficiency,the electroporation conditions were optimized.The positive transformant was obtained after phenotype screening and PCR identification.Inducible expression of the recombinant P.pastoris GS115-GT was conducted.Target protein detected by SDS-PAGE verified β-1,4-galactosyltransferase gene was expressed solubly in P.pastoris GS115.The specific activity of β-1,4-galactosyltransferase after optimization of fermentation conditions for genetically engineered microorganim was 30.909 U/ml increased by 88.47% compared to that before optimization,which was only 16.40 U/ml.

关 键 词：Β-1 4-半乳糖基转移酶 电转化 诱导表达 发酵条件 优化 

分 类 号：TQ920.1[轻工技术与工程—发酵工程]