玉米谷氨酰胺酶基因在毕赤酵母中表达条件的优化  

Optimization of Expression Conditions of Zea mays Transglutaminase Gene in Pichia pastoris

作　　者：韩秀娥 秦兰霞 邵红 任浩威 Han Xiue;Qin Lanxia;Shao Hong;Ren Haowei(College of Food Science,Northeast Agricultural University,Harbin 150030)

机构地区：[1]东北农业大学食品学院,哈尔滨150030

出　　处：《中国食品学报》2022年第4期76-82,共7页Journal of Chinese Institute Of Food Science and Technology

基　　金：黑龙江省自然科学基金项目(LH2019C030)。

摘　　要：为确定玉米谷氨酰胺转氨酶基因在毕赤酵母中的最佳诱导表达条件,将构建好的重组表达载体pPIC9K-TGZ经Sac I线性化处理,电转入巴斯德毕赤酵母GS115,利用抗生素Zeocin筛选阳性克隆,聚合酶链式反应扩增验证,甲醇诱导表达。通过正交试验确定工程菌最佳表达条件:表达培养基BMMY,诱导表达时间96 h,初始表达pH值6.0,菌体起始诱导OD_(600nm)值4.0,诱导温度24℃,甲醇添加量0.6%(体积分数)、甲醇流加时间间隔12 h/次,诱导48 h后每24 h添加1 g/L甘油。采用优化的条件得到培养基上清的最高酶活达7.51 U/mL。In order to determine the optimal inducible expression conditions of zea mays transglutaminase gene in Pichia pastoris, pPIC9 K-TGZ was introduced into the component P. pastoris GS115 strains by the electroporation method,screening positive clones with antibiotics, PCR amplification verification, IPTG induced expression, optimize the best expression conditions. In this study, the optimal expression conditions of engineering bacteria were determined by orthogonal experiment. The expression medium was BMMY and the induced expression time was 96 h, initial expression of pH 6.0,the initial induction OD_(600 nm)of the bacteria was 4.0;the induction temperature was 24 ℃;the methanol content was 0.6%(volume fraction);methanol flow was added once/12 h;glycerol of 1 g/L was added every 24 hours after 48 h induction.The maximum enzyme activity of medium supernatant under optimized conditions was 7.51 U/mL.

关 键 词：玉米 谷氨酰胺转氨酶 毕赤酵母 表达 表达条件优化 

分 类 号：Q78[生物学—分子生物学] TS201.25[轻工技术与工程—食品科学]