Chen J T, Chen M C, Chen L L, Chu W S
Food Industry Research and Development Institute, P.O. Box 246, Hsinchu 30099, Taiwan, Republic of China.
Biotechnol Appl Biochem. 2001 Jun;33(3):189-99. doi: 10.1042/ba20010003.
An amylopullulanase gene (apuTS) from Bacillus stearothermophilus TS-23 was cloned and characterized. apuTS consisted of an open reading frame of 6054 bp encoding a protein of 2018 amino acids with a calculated M(r) of 223811. The deduced amino acid sequence revealed four highly conserved regions that are common among amylolytic enzymes. In the C-terminal region, a six-amino-acid sequence (Pro-Gly-Ser-Gly-Thr-Thr) is repeated nine times. It shared the highest degree of homology with the amylopullulanase of Bacillus sp. XAL601. The enzyme also had moderate homology with amylopullulanases from thermophilic anaerobic bacteria. Low levels of homology were observed between the ApuTS of B. stearothermophilus TS-23 and amylopullulanases of Pyrococcus abyssi Orsay, P. furiosus and Bacillus sp. KSM1378. When the intact coding region of apuTS was expressed in Escherichia coli under the control of the lac promoter, the product was degenerate, as revealed by amylase activity staining after SDS/PAGE. The largest active polypeptide had an M(r) of about 220000, while the smallest one had an M(r) of about 105000. Upstream of the apuTS gene, a gene orfX was fortuitously cloned. The putative OrfX protein was weakly related to the myosin heavy chain. It was predicted to contain a central, 179-residue-long, coiled-coil domain.
克隆并鉴定了嗜热脂肪芽孢杆菌TS-23的支链淀粉酶基因（apuTS）。apuTS由一个6054 bp的开放阅读框组成，编码一个含2018个氨基酸的蛋白质，计算分子量为223811。推导的氨基酸序列显示出在淀粉分解酶中常见的四个高度保守区域。在C端区域，一个六氨基酸序列（Pro-Gly-Ser-Gly-Thr-Thr）重复了九次。它与芽孢杆菌属XAL601的支链淀粉酶具有最高程度的同源性。该酶与嗜热厌氧细菌的支链淀粉酶也有中等程度的同源性。嗜热脂肪芽孢杆菌TS-23的ApuTS与深渊热球菌奥尔赛、激烈火球菌和芽孢杆菌属KSM1378的支链淀粉酶之间的同源性较低。当apuTS的完整编码区在乳糖启动子控制下在大肠杆菌中表达时，如SDS/PAGE后的淀粉酶活性染色所示，产物是退化的。最大的活性多肽分子量约为220000，而最小的约为105000。在apuTS基因上游，偶然克隆了一个orfX基因。推测的OrfX蛋白与肌球蛋白重链有弱相关性。预计它含有一个中心的、179个残基长的卷曲螺旋结构域。
