Sulfolobus acidocaldarius ATCC 33909麦芽寡糖基海藻糖合成酶在Bacillus subtilis中的重组表达和发酵优化

Recombinant Expression and Fermentation Optimization of Sulfolobus acidocaldarius ATCC 33909 Maltooligosyltrehalose Synthase in Bacillus subtilis

为实现Sulfolobus acidocaldarius ATCC 33909来源的麦芽寡糖基海藻糖合成酶(MTSase)基因tre Y在枯草芽孢杆菌(Bacillus subtilis)中的重组表达,以质粒p ET-24a(+)-tre Y为模板PCR扩增得到目的基因,并与表达载体pHY300PLK连接,转入表达宿主Bacillus subtilis CCTCC M 2016536中,重组菌在TB培养基中培养48 h后MTSase酶活达到17.5 U/m L;在此基础上对重组菌发酵条件进行优化,通过单因素实验(氮源种类、氮源复配、氮源浓度、碳源种类、葡萄糖浓度、初始pH、诱导温度)和正交实验(氮源浓度、葡萄糖浓度、初始pH、诱导温度)确定其摇瓶发酵产酶的最适培养基和培养条件为:氮源(工业蛋白胨∶棉籽粉=3∶1)48.0 g/L、葡萄糖为10.0 g/L、培养基初始pH为7.0,最适培养温度为30℃;在此条件下,MTSase的酶活可达41.5 U/m L,是优化前的2.4倍。

To obtain the recombinant expression of gene tre Y for maltooligosyltrehalose synthase（MTSase）from Sulfolobusacidocaldarius ATCC 33909 in Bacillus subtilis,the target gene was PCR-amplified using plasmid p ET-24a（＋）-tre Y as template,andligated with the expression vector pHY300 PLK,then transformed into the expression host Bacillus subtilis CCTCC M 2016536. The activityof MTSasereached 17.5 U/m L after cultivated in TB culture for 48 h. By single factor test（nitrogen source,nitrogen proportion,nitrogenconcentration,carbon source,glucose concentration,initial pH,and induction temperature）and orthogonal test（nitrogen concentration,glucose concentration,initial pH,and induction temperature）,the optimal fermentation condition was determined as ：48.0 g/L of nitrogensource（industrial peptone∶cottonseed powder=3∶1）,10.0 g/L of glucose,initial pH of medium=7.0,and the optimal temperature was 30℃. Under optimal conditions,the production of MTSase reached 41.5 U/m L,which was 2.4 times of that before optimization.