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

排泄载体大肠杆菌产胞外酶的工程研究

基本信息

批准号：
02650711
负责人：
YAMANE Tsuneo
金额：
$ 1.15万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

项目摘要

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.

1.分泌型重组大肠杆菌的补料分批培养研究了培养基组成和培养条件对嗜碱芽孢杆菌分泌青霉素酶的影响。大肠杆菌中进行了研究。LB培养基+甘油促进了酶的分泌。接下来，在3L生物反应器中进行两阶段补料分批培养，其中供应足够的LB +甘油直至约1000 g。20 gDC/L的细胞浓度，以尽可能抑制细胞裂解，然后控制LB +甘油的进料。甘油的有限补料对胞外酶的产生是有效的，其过量补料使细胞生长丰富，酶的形成较少。当甘油比供给速率为0.10[g（g DC）·h^ ]时，酶活最高<-1><-1>，是分批培养的20倍，产酶能力[（Unit）L·<-1>h^<-1>]提高了5倍。第二生长期的分泌量大于90%。即使在最好的补料分批培养中也观察到少量细胞溶解，这意味着需要进一步改善Kil基因的弱表达。2.分泌载体的改良（堀越幸）为了提高青霉素酶基因的分泌能力，构建了同时携带青霉素酶基因启动子和tac启动子的分泌载体pEAP 85。将嗜碱芽孢杆菌的环糊精合成酶（CGTase）基因导入载体质粒，并对其分泌产物进行了研究。CGTase在重组E.大肠杆菌中的CGTase分泌到培养上清中。