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

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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.