摘要
应用PCR从大肠杆菌基因组中扩增L-阿拉伯糖异构酶基因,用EcoR I和Not I双酶切将其克隆进P.pastoris表达载体,获得重组表达载体pGAP9K-L-ai。通过电转法将pGAP9K—L-ai转化毕赤酵母GS115,筛选高G418抗性和高表达L-阿拉伯糖异构酶的重组工程菌。用葡萄糖作为碳源在摇瓶中发酵48 h,表达重组L-ai 53 mg/L。用毕赤酵母的GAP启动子调控表达的重组L-ai具有异构D-半乳糖生成D-塔格糖的生物学活性。
L-arabinose isomerase gene was amplified from E. coli by PCR technique according to the L-ai sequence and cloned directly into plasmid pGAP9K to obtain P. pastoris expression vector pGAP-L-ai which was then transformed into the P. pastoris GS115 by electroporation method. A high gene copied and high L-ai expressed strain was used as engineer- ing strain and selected by replicating the transformants on G418 containing plates and expressing them. After two days fer- mentation in shaking flask using glucose as carbon source, 53 mg/L of recombinant L-ai was secreted in the medium. The recombinant L-arabinose isomerase showed its biological activity in changing D-galactose to D-tagatose.
出处
《工业微生物》
CAS
CSCD
2014年第1期51-54,共4页
Industrial Microbiology
基金
中央级公益性科研院所基本科研业务费专项资金资助项目(ITBB120503)
