D-氨基酸氧化酶在不同毕赤酵母宿主菌中的表达比较  

Compared D-amino Acid Oxidase Expression in Different Pichia pastoris Host Strains

作　　者：冯美卿[1] 黄海[1] 史训龙[1] 余志良[2] 袁中一[2] 周珮[1] 

机构地区：[1]复旦大学药学院 [2]中国科学院上海生命科学院生物化学与细胞生物学研究所,上海200031

出　　处：《生物工程学报》2004年第4期572-577,共6页Chinese Journal of Biotechnology

摘　　要：D 氨基酸氧化酶 (DAAO)在转化头孢菌素C生产 7 ACA和转化DL 氨基酸制备α 酮酸和L 氨基酸上起着重要的作用。采用DNA操作技术 ,将来源于三角酵母的DAAO基因连接至表达载体pPIC3 5K上 ,再将表达质粒pPIC3 5K DAAO分别整合P .pastoris的宿主细胞KM71和GS1 1 5 ,经筛选获得阳性重组菌PDK1 3(MutS)和PD2 7(Mut+ )。重点对两种突变菌的表达条件进行了比较。结果显示 :PDK1 3(MutS)株比PD2 7(Mut+ )株消耗甲醇慢、诱导时间长 ,但对通气量要求低、表达水平高 ,摇瓶活力分别达到 2 70 0和 2 5 0 0IU L ,1 4L发酵罐内活力分别达到 1 0 1 4 0和 84 6 3IU L。初步探索了DAAO对DL 苯丙氨酸的拆分 ,结果显示基因工程菌表达的DAAO具有良好的转化DL 苯丙氨酸制备苯丙酮酸和LTo compare the DAAO expression level in different Pichia pastoris host strains, the gene encoding DAAO from Trigonopsis variabilis was cloned into plasmid pPIC3 5k and then transformed into P. pastoris GS115 and KM71 respectively. The positive transformants PDK13( Mut S) and PD27( Mut +) were obtained by PCR analysis. Their optimal and different expression conditions were investigated. To compare with PD27, PDK13 was determined to poss a slower consumption of methanol, a longer induction time, a lower oxygen request and apparently higher expression of DAAO . The highest expression levels were reached up to 2700, 2500 IU/L in shaking flask and 10140, 8463 5 IU/L in fermentor respectively. The over expression of DAAO can meet its large demand for production of 7 ACA, α keto acid and L amino acid. In addition, the phenylpyruvate and L phenylalanine were obtained by crude DAAO reacting with DL phenylalanine at 37℃ for 3h.

关 键 词：D-氨基酸氧化酶 甲醇酵母 不同宿主菌 表达比较 DL-苯丙氨酸 拆分 

分 类 号：Q55[生物学—生物化学]