摘要
将编码甘油单-二酰酯脂肪酶(MDGL)的基因mdlA插入到分泌表达质粒pPIC9K中,通过电激将线性化的重组质粒整合到毕赤酵母(Pichia pastoris)GS115中,筛选出H is+Mut+表型菌株,进一步用G418筛选获得高拷贝转化子,并用PCR方法鉴定。诱导培养后,SDS-PAGE表明MDGL在毕赤酵母中得到有效表达。表达产物在温度40℃,pH7.5具有最高活性,其发酵液酶活可达到325U/mL,以橄榄油为底物时没有检测到活性。表达产物与甘油三酰酯脂肪酶共同作用时产生的脂肪酸量比甘油三酰酯脂肪酶单独作用提高了93.5%。
The mono- and diacylglycerol lipase （MDGL） -encoding gene （mdbt） was inserted into methanolinducible expression vector pPIC9K. The linearized recombinant plasmid was transformed into chromosome of Pichia pastoris GS115 strain by electroporation. Some high-copy transformants （ His ^＋ Mut^ ＋ ） were picked up by G418 and conformed by PCR. SDS- PAGE analysis indicated efficient expression of recombinant MDGL Some enzymatic properties of the recombinant MDGL were also determined. Tbe activity of the recombinant MDGL was up to 325U/mL under the optimal conditions and no activity was detected towards olive oil. The amount of fatty acid produced by the catalysis of recombinant MDGL and triacylglycerol lipase had a increase of 93.5% over triacylglycerol lipase lonely.
出处
《微生物学通报》
CAS
CSCD
北大核心
2006年第1期18-23,共6页
Microbiology China
基金
北京市生物加工过程重点实验室开放项目(No.SYS100100421)
关键词
甘油单-二酰酯脂肪酶
毕赤酵母
表达
Mono- and diacylglycerol lipase, Pichia pastoris, Expression
