Fukusumi S, Kamizono A, Horinouchi S, Beppu T
Department of Agricultural Chemistry, University of Tokyo, Japan.
Eur J Biochem. 1988 May 16;174(1):15-21. doi: 10.1111/j.1432-1033.1988.tb14056.x.
A highly heat-stable amylase gene from an obligately anaerobic and extremely thermophilic bacterium, Dictyoglomus thermophilum, was cloned and expressed in Escherichia coli. The nucleotide sequence of the amylase gene predicts a 686-amino-acid protein of relative molecular mass 81,200, which is consistent with that determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the purified enzyme. The NH2-terminal sequence determined using the enzyme purified from E. coli cells corresponds precisely to that predicted from the nucleotide sequence, except for the absence of the NH2-terminal methionine in the mature protein. When the amylase gene was expressed in E. coli cells, the enzyme was localized in the cytoplasmic fraction; this is probably explained by the absence of the signal sequence for secretion. By using the amylase purified from the E. coli transformant, some enzymatic properties, such as optimum pH, optimum temperature, pH-stability and heat-stability, were examined. The amylase was found to be a highly liquefying-type.
从专性厌氧极端嗜热菌嗜热网柱菌（Dictyoglomus thermophilum）中克隆出一个高度耐热的淀粉酶基因，并在大肠杆菌中进行表达。淀粉酶基因的核苷酸序列预测其编码一个由686个氨基酸组成、相对分子质量为81200的蛋白质，这与通过纯化酶的十二烷基硫酸钠/聚丙烯酰胺凝胶电泳测定的结果一致。使用从大肠杆菌细胞中纯化的酶测定的NH2末端序列，除了成熟蛋白中不存在NH2末端甲硫氨酸外，与从核苷酸序列预测的结果完全一致。当淀粉酶基因在大肠杆菌细胞中表达时，该酶定位于细胞质部分；这可能是由于缺乏分泌信号序列所致。通过使用从大肠杆菌转化体中纯化的淀粉酶，研究了一些酶学性质，如最适pH、最适温度、pH稳定性和热稳定性。发现该淀粉酶是一种高度液化型的。
