豆豉纤溶酶在枯草杆菌WB800中的高水平表达

一个来源于豆豉产生菌DC12的新的纤溶酶基因序列被克隆测序.为了实现在枯草杆菌中高水平表达豆豉纤溶酶,将来源于枯草杆菌168的重叠启动子P43序列以及转录终止信号序列通过重叠延伸融合,将融合序列克隆到大肠杆菌-枯草杆菌穿梭载体pBE3中,构建了pBEP43载体.将豆豉纤溶酶基因插入到载体pBEP43后转化枯草杆菌WB800.结果表明,在P43启动子驱动下,豆豉纤溶酶基因在重组菌中的对数生长期和平衡期均获得了表达且分泌到培养基中.重组菌经培养后上清液中的纤溶酶活性最高达1270U/mL,是豆豉纤溶酶基因在自身启动子驱动下表达量的1.87倍和野生菌株枯草杆菌DC12表达量的4.1倍.图5参16

High-level Expression of Douchi Fibrinolytic Enzyme (DFE) in Bacillus subtilis WB800

A novel fibrinolytic enzyme gene was cloned from a Douchi producing strain, Bacillus subtilis DC12. To efficiently expressed Douchi fibrinolytic enzyme (DFE) in B. subtilis, the overlapping promoter P43 sequence cloned from B. subtilis 168 and a B. subtilis terminator were fused by overlapping PCR, and inserted into pBE3 to yield a novel vector, pBEP43. Then the encoding DFE gene was cloned into pBEP43 and expressed in B. subtilis WB800 under the control of promoter P43. Results showed that DFE was efficiently expressed during the exponential growth and stationary time, and secreted into the medium. The maximum fibrinolytic activity level of 1 270 U/mL was 1.87-fold to its expression mediated by its own promoter in B. subtilis WB800, 4.1-fold to that of the wild-type strain B. subtilis DC12. Fig 5, Ref 16