Wei D, Parulekar S J, Stark B C, Weigand W A
Department of Chemical Engineering, Illinois Institute of Technology, Chicago, Illinois 60616, USA.
Biotechnol Bioeng. 1989 Mar;33(8):1010-20. doi: 10.1002/bit.260330810.
Cell growth and enzyme (alpha-amylase) production characteristics of Bacillus subtilis TN106 containing the recombinant plasmid pAT5 are investigated in batch and continuous cultures using a defined medium with glucose as the limiting nutrient. The batch culture studies demonstrate that the recombinant plasmid, reported earlier(1) to be stably maintained in the host, suffers from segregational and structural instabilities. The structural instability of this strain occurred during culture storage and can be eliminated in bioreactor experiments by using a modified inoculum preparation procedure. Such elimination allows an unbiased investigation of segregational instability via continuous culture studies. Such studies conducted with this fast growing microorganism, in the absence of antibiotic selection pressure, indicate a very efficient glucose utilization (very low residual glucose concentrations) over a wide range of dilution rates (0.16 h(-1) - 0.94 h(-1)). The nearly time-invariant and low residual glucose concentrations at each such dilution rate enable convenient estimation of growth parameters of the host and recombinant cells and frequency of segregational instability from transients in the resulting mixed cultures. The specific alpha-amylase activity exhibits an inverse relationship to the specific growth rate of recombinant cells. The growth of recombinant cells is not affected by the presence of antibiotic (kanamycin). The growth advantage of host cells over recombinant cells diminishes with increasing dilution rate.
使用以葡萄糖作为限制性营养物质的限定培养基，在分批培养和连续培养中研究了含有重组质粒pAT5的枯草芽孢杆菌TN106的细胞生长和酶（α-淀粉酶）产生特性。分批培养研究表明，先前报道（1）可在宿主中稳定维持的重组质粒存在分离和结构不稳定性。该菌株的结构不稳定性在培养物保存期间发生，并且可以通过使用改良的接种体制备程序在生物反应器实验中消除。这种消除使得能够通过连续培养研究对分离不稳定性进行无偏差的研究。在没有抗生素选择压力的情况下，对这种快速生长的微生物进行的此类研究表明，在很宽的稀释率范围（0.16 h(-1)-0.94 h(-1)）内，葡萄糖利用效率非常高（残余葡萄糖浓度非常低）。在每个这样的稀释率下，几乎随时间不变且低的残余葡萄糖浓度使得能够方便地从所得混合培养物中的瞬变情况估计宿主细胞和重组细胞的生长参数以及分离不稳定性的频率。α-淀粉酶的比活性与重组细胞的比生长速率呈反比关系。重组细胞的生长不受抗生素（卡那霉素）存在的影响。宿主细胞相对于重组细胞的生长优势随着稀释率的增加而减弱。
