Riethdorf S, Ulrich A, Völker U, Hecker M
Sektion Biologie, Ernst-Moritz-Arndt-Universität, Greifswald.
Z Naturforsch C J Biosci. 1990 Mar-Apr;45(3-4):240-4. doi: 10.1515/znc-1990-3-416.
The beta-glucanase gene (bgl) from Bacillus amyloliquefaciens was expressed in E. coli CSH 55 under the control of the PR promoter of phage lambda that is repressed by the thermosensitive repressor C1857. Production of beta-glucanase was drastically stimulated by a temperature shift to 42 degrees C. This overexpression of the bgl gene (about 20% of the total cellular protein) led to an almost complete excretion of the otherwise periplasmic protein into the extracellular medium, beta-glucanase accounted for more than 50% of the extracellular proteins. Col E 1 related plasmid (pEG 1) are amplified in E. coli relA strains in response to an amino acid limitation leading to a 10-fold increase in the activity of plasmid encoded genes. In this work we intended to maximize the expression of the bgl gene by a concerted action of a plasmid amplification and temperature induction. Surprisingly we could not increase the beta-glucanase production above the level reached by plasmid amplification or temperature induction alone. The reasons for this unexpected result will be discussed. Under all conditions tested the expression of the bgl gene was much lower in the E. coli relA strain NF 162 than in E. coli CSH 55; the low beta-glucanase production was accompanied by a reduced excretion rate of the enzyme.
解淀粉芽孢杆菌的β-葡聚糖酶基因（bgl）在噬菌体λ的PR启动子控制下于大肠杆菌CSH 55中表达，该启动子受温度敏感阻遏物C1857的抑制。温度转移至42℃可极大地刺激β-葡聚糖酶的产生。bgl基因的这种过表达（约占细胞总蛋白的20%）导致原本位于周质的蛋白几乎完全分泌到细胞外培养基中，β-葡聚糖酶占细胞外蛋白的50%以上。与Col E 1相关的质粒（pEG 1）在大肠杆菌relA菌株中因氨基酸限制而扩增，导致质粒编码基因的活性增加10倍。在这项工作中，我们打算通过质粒扩增和温度诱导的协同作用使bgl基因的表达最大化。令人惊讶的是，我们无法将β-葡聚糖酶的产量提高到单独通过质粒扩增或温度诱导所达到的水平之上。将讨论这一意外结果的原因。在所有测试条件下，bgl基因在大肠杆菌relA菌株NF 162中的表达远低于在大肠杆菌CSH 55中的表达；β-葡聚糖酶产量低伴随着该酶分泌速率的降低。
