Sakamoto S, Terada I, Lee Y C, Uehara K, Matsuzawa H, Iijima M
Life Science Research Laboratory, Japan Tobacco Inc., Yokohama.
Appl Microbiol Biotechnol. 1996 Mar;45(1-2):94-101. doi: 10.1007/s002530050654.
The DNA sequence encoding Thermus protease aqualysin I was inserted downstream from a bacteriophage T7 promoter in an expression vector. In the T7 expression system, using a strain lacking an F' episome, aqualysin I was produced in soluble form without chemical induction. The deletions of part (30 amino acid residues) or all (105 residues) of the C-terminal pro-sequence from the C terminus significantly affected both cellular growth and the production of the enzyme. Complete deletion adversely affected both. In contrast, the 30-residue deletion markedly improved productivity by approximately four times compared to non-deletion, and shortened the time needed for the activation of a precursor to active enzyme. The concentration of inducer isopropyl beta-D-thiogalactopyrano-side (IPTG) was varied to examine its effects, and it was found that a low concentration of IPTG improved aqualysin I production. To avoid the inhibitory effects of acetic acid accumulation in the culture medium, the use of other carbon sources besides glucose was examined. When cells were cultivated with glycerol, the acetic acid level remained relatively low, and both good cellular growth and a high level of production were attained. The aqualysin I productivity for a fed-batch culture using two carbon sources, glucose and glycerol, reached more than 150 kU/ml enzymatically active aqualysin I.
将编码嗜热栖热菌蛋白酶水栖溶素I的DNA序列插入到表达载体中噬菌体T7启动子的下游。在T7表达系统中，使用缺乏F'附加体的菌株，水栖溶素I以可溶形式产生，无需化学诱导。从C末端缺失部分（30个氨基酸残基）或全部（105个残基）的C末端前序列，对细胞生长和酶的产生均有显著影响。完全缺失对两者均有不利影响。相比之下，与未缺失相比，30个残基的缺失使生产率显著提高了约四倍，并缩短了前体激活为活性酶所需的时间。改变诱导剂异丙基-β-D-硫代半乳糖苷（IPTG）的浓度以检查其效果，发现低浓度的IPTG可提高水栖溶素I的产量。为避免培养基中乙酸积累的抑制作用，研究了除葡萄糖外其他碳源的使用。当用甘油培养细胞时，乙酸水平保持相对较低，细胞生长良好且产量较高。使用葡萄糖和甘油两种碳源进行补料分批培养时，水栖溶素I的生产率达到了超过150 kU/ml的酶活性水栖溶素I。
