Ikawa K, Araki H, Tsujino Y, Hayashi Y, Igarashi K, Hatada Y, Hagihara H, Ozawa T, Ozaki K, Kobayashi T, Ito S
Tochigi Research Laboratories of Kao Corporation, Japan.
Biosci Biotechnol Biochem. 1998 Sep;62(9):1720-5. doi: 10.1271/bbb.62.1720.
We have constructed a new excretion vector, pHSP64, to develop a hyperexcretion system for Bacillus subtilis [Sumitomo et al., Biosci. Biotech. Biochem., 59, 2172-2175 (1995)]. The structural gene for a novel liquefying semi-alkaline alpha-amylase from the alkaliphilic Bacillus sp. KSM-1378 was amplified by PCR. It was cloned into a SalI-SmaI site of pHSP64 and the recombinant plasmid obtained was introduced into B. subtilis. The transformed B. subtilis hyperproduced the alpha-amylase activity extracellularly, corresponding to approximately 1.0 g (5 x 10(6) units) per liter of an optimized liquid culture. The recombinant enzyme was purified to homogeneity by a simple purification procedure with very high yield. No significant differences in physiochemical and catalytic properties were observed between the recombinant enzyme and the native enzyme produced by Bacillus sp. KSM-1378. The enzymatic properties of the recombinant enzyme were further examined with respect to the responses to various metal ions. The recombinant enzyme could easily be crystallized at room temperature within one day in a buffered solution of 10% (w/v) ammonium sulfate (pH 6.5).
我们构建了一种新的分泌载体pHSP64，以开发枯草芽孢杆菌的高效分泌系统[Sumitomo等人，《生物科学、生物技术与生物化学》，59，2172 - 2175（1995）]。通过PCR扩增了嗜碱芽孢杆菌KSM - 1378的一种新型液化半碱性α -淀粉酶的结构基因。将其克隆到pHSP64的SalI - SmaI位点，得到的重组质粒导入枯草芽孢杆菌。转化后的枯草芽孢杆菌在细胞外过量产生α -淀粉酶活性，在优化的液体培养中，每升约产生1.0克（5×10⁶单位）。通过简单的纯化程序，以非常高的产率将重组酶纯化至同质。重组酶与嗜碱芽孢杆菌KSM - 1378产生的天然酶在物理化学和催化特性上未观察到显著差异。进一步研究了重组酶对各种金属离子的响应的酶学性质。重组酶在室温下，在10%（w/v）硫酸铵（pH 6.5）的缓冲溶液中，一天内即可轻松结晶。
