Hemilä H, Pokkinen M, Palva I
Institute of Biotechnology, University of Helsinki, Finland.
J Biotechnol. 1992 Nov;26(2-3):245-56. doi: 10.1016/0168-1656(92)90010-7.
Bacillus subtilis has been considered a promising host for the production of foreign proteins. However, proteases released by the host organism can often cause rapid breakdown of secreted heterologous proteins. Here we report that the addition of 6% glucose and 100 mM potassium phosphate to the growth medium significantly reduces the degradation of E. coli TEM beta-lactamase secreted from B. subtilis, when applying an expression system based on B. amyloliquefaciens alpha-amylase. The yield of beta-lactamase was increased 10-20-fold when compared to the yield in Luria medium. The promoter of B. amyloliquefaciens alpha-amylase gene is repressed by glucose. However, here we show that the repression does not take place in a multicopy plasmid, thus enabling our approach to efficiently reduce the protease action by catabolite repression. We have also studied the role of pH and temperature on the beta-lactamase production in laboratory scale bioreactors. Low temperature and low pH are both favorable for a high level beta-lactamase production by the high copy plasmid construction.
枯草芽孢杆菌被认为是生产外源蛋白的理想宿主。然而，宿主生物体释放的蛋白酶常常会导致分泌的异源蛋白迅速降解。在此我们报告，当应用基于解淀粉芽孢杆菌α-淀粉酶的表达系统时，在生长培养基中添加6%葡萄糖和100 mM磷酸钾可显著减少枯草芽孢杆菌分泌的大肠杆菌TEMβ-内酰胺酶的降解。与在Luria培养基中的产量相比，β-内酰胺酶的产量提高了10至20倍。解淀粉芽孢杆菌α-淀粉酶基因的启动子受葡萄糖抑制。然而，在此我们表明这种抑制在多拷贝质粒中不会发生，因此我们的方法能够通过分解代谢物阻遏有效地减少蛋白酶的作用。我们还在实验室规模的生物反应器中研究了pH和温度对β-内酰胺酶产生的作用。低温和低pH都有利于通过高拷贝质粒构建实现高水平的β-内酰胺酶产生。
