Engineering Study on Extracellular Enzyme Production by Escherichia coli Harboring an Excretion Vector

1. 研究了培养基组成和培养条件对携带分泌载体的大肠杆菌分泌嗜碱芽孢杆菌青霉酶的影响。LB培养基+甘油促进酶的分泌。接下来，在3L生物反应器中进行两段补料分批培养，其中提供足够的LB +甘油（细胞浓度高达约20 gDC/L）以尽可能地抑制细胞裂解，然后控制LB +甘油的补料。有限的甘油摄食对细胞外酶的产生是有效的，过量的甘油摄食使细胞生长旺盛，酶的形成较少。当甘油比供率为0.10[g(g DC) ^<-1>h^<-1>]时酶活性最高，是批量培养的20倍，酶产率[（单位）L ^<-1>h^<-1>]提高了5倍。第二生长期分泌量大于90%。即使在最好的补料批培养中也观察到少量的细胞裂解，这意味着需要进一步改善Kil基因的弱表达。以提高kil基因分泌载体的表达能力为目的，构建了同时携带青霉酶基因启动子和tac启动子的分泌载体peap85。将嗜碱性芽孢杆菌的环糊精合成酶（CGTase）基因导入载体质粒，研究其分泌量。重组大肠杆菌中高表达CGTase，大部分CGTase被分泌到培养上清液中。

1. Fed-batch Culture of Recombinant Escherichia coli Carrying a Secretion Vector (Tsuneo YAMANE) Effects of medium composition and culture condition on the secretion of penicillinase of alkalophilic Bacillus sp. by E. coli havoring a secretion vector were studied. LB medium + glycerol enhanced the enzyme secretion. Next, two-stage fed-batch cultures were in a 3L bioreactor where enough LB + glycerol were supplied upto ca. 20 gDC/L of cell concentration in order to suppress cell lysis as much as possible, followed by controlled feeding of LB + glycerol. The limited feeding of glycerol was effective for the extracellular enzyme production and its overfeeding gave abundant cell growth with lesser formation of the enzyme. The highest enzyme activity was obtained when glycerol's specific supply rate was 0.10[g(g DC) ^<-1>h^<-1>], which was 20 times as much as that of a batch culture and the enzyme productivity [(Unit)L ^<-1>h^<-1>] was raised 5 -fold. Secretion in the second growth phase was more than 90%. A little cell lysis was observed even in the best fed-batch culture, implying further improvements in the weak expression of Kil gene are required.2. Improvement of Secretion Vector (Koki Horikoshi)A secretion vector, pEAP 85, which carried both promoter of penicillinase gene and tac promoter, was constructed with the aim of increasing ability of secretion vector of kil gene. Cyclodextrine synthetase (CGTase) gene of an alkalophilic Bacillus sp. was introduced to the vector plasmid, and its secretory production was studied. CGTase was highly expressed in the recombinant E. coli and most CGTase was secreted into the culture supernatant.