Inoue Yasushi, Ishii Keiko, Tomita Tetsuji, Yatake Tsuneya, Fukui Fumio
Showa Sangyo Co, Ltd, Tsukubashi, Ibaraki, Japan.
Biosci Biotechnol Biochem. 2002 Sep;66(9):1835-43. doi: 10.1271/bbb.66.1835.
A bacterial trehalose phosphorylase (TPase; EC 2.4.1.64) was purified from the culture supernatant of Bacillus stearothermophilus SK-1 to apparent homogeneity, and some properties were investigated. Furthermore, a gene from SK-1 responsible for the TPase was cloned by Southern hybridization with a degenerate oligonucleotide probe synthesized on the basis of the N-terminal sequence of the purified enzyme. The Mr of the enzyme was estimated to be 150,000 by gel filtration and 83,000 by SDS-PAGE, so the enzyme is likely to be a homodimer. The enzyme had optimum activity at pH 7.0-8.0 or nearby and the optimum temperature was about 75 degrees C. The deduced amino acid sequence of the SK-1 TPase encodes a theoretical protein with a Mr of 87,950. Alignment of amino acid sequences with a maltose phosphorylase from Lactobacillus brevis the crystal structure and active site of which had been analyzed suggested that these two phosphorylases evolved from a common ancestor. The Escherichia coli cells harboring the plasmid containing the cloned TPase gene had about 100 times the activity of SK-1.
从嗜热脂肪芽孢杆菌SK-1的培养上清液中纯化出一种细菌海藻糖磷酸化酶（TPase；EC 2.4.1.64），使其达到表观均一性，并对其一些性质进行了研究。此外，通过与基于纯化酶N端序列合成的简并寡核苷酸探针进行Southern杂交，克隆了SK-1中负责TPase的基因。通过凝胶过滤法估计该酶的Mr为150,000，通过SDS-PAGE法估计为83,000，因此该酶可能是同型二聚体。该酶在pH 7.0 - 8.0或附近具有最佳活性，最佳温度约为75℃。SK-1 TPase推导的氨基酸序列编码一种理论蛋白，其Mr为87,950。将氨基酸序列与短乳杆菌的麦芽糖磷酸化酶进行比对，该麦芽糖磷酸化酶的晶体结构和活性位点已被分析，结果表明这两种磷酸化酶由共同的祖先进化而来。携带含有克隆TPase基因质粒的大肠杆菌细胞的活性约为SK-1的100倍。
