Matsui T, Sato H, Sato S, Mukataka S, Takahashi J
Institute of Applied Biochemistry, University of Tsukuba, Ibaraki, Japan.
Agric Biol Chem. 1990 Mar;54(3):619-24.
Effects of nutritional conditions and insertion direction of the tryptophan synthase (TSase) gene into a plasmid vector on the plasmid stability and the production of TSase in high cell concentration cultures were examined using recombinant Escherichia coli (E. coli K12 IFO 3301) harboring pBR322trpAB(1) (the TSase gene was inserted at the EcoRI site of pBR322 in the clockwise direction) and pBR322trpAB(2) (counterclockwise direction). As to the effects of the insertion direction, the cells harboring pBR322trpAB(2) were slightly lower in the growth rate and the plasmid stability than those harboring pBR322trpAB(1). However, the former was higher in the productivity of TSase and the final cell concentration attained than the latter. On the other hand, the addition of organic nutrients, especially yeast extract, to TK-25 medium was very effective to improve the plasmid stability. Among the components of yeast extract, L-glutamic acid was found to be effective to improve both the plasmid stability and the production of TSase. When 1 g/l of L-glutamic acid was added to TK-25 medium, a mineral synthetic medium developed for a high concentration culture, 115g (dry basis)/l of recombinant cells were obtained in 14 hr and the expression of TSase was maintained at 240-300 U/mg-protein during the cultivation.
利用携带pBR322trpAB(1)（色氨酸合成酶基因以顺时针方向插入pBR322的EcoRI位点）和pBR322trpAB(2)（逆时针方向）的重组大肠杆菌（大肠杆菌K12 IFO 3301），研究了营养条件以及色氨酸合成酶（TSase）基因插入质粒载体的方向对高细胞浓度培养物中质粒稳定性和TSase产量的影响。关于插入方向的影响，携带pBR322trpAB(2)的细胞在生长速率和质粒稳定性方面略低于携带pBR322trpAB(1)的细胞。然而，前者的TSase生产率和最终达到的细胞浓度高于后者。另一方面，向TK - 25培养基中添加有机营养物，尤其是酵母提取物，对提高质粒稳定性非常有效。在酵母提取物的成分中，发现L - 谷氨酸对提高质粒稳定性和TSase产量均有效。当向为高浓度培养开发的矿物合成培养基TK - 25中添加1 g/l的L - 谷氨酸时，在14小时内获得了115 g（干基）/l的重组细胞，并且在培养过程中TSase的表达维持在240 - 300 U/mg蛋白质。
