Kashiwabara S, Matsuki Y, Kishimoto T, Suzuki Y
Department of Applied Biochemistry, Kyoto Prefectural University, Japan.
Biosci Biotechnol Biochem. 1998 Jun;62(6):1093-102. doi: 10.1271/bbb.62.1093.
The gene that coded for a cellular oligo-1,6-glucosidase (dextrin 6-alpha-D-glucanohydrolase, EC 3.2.1.10) in Bacillus flavocaldarius KP1228 (FERM-P9542) cells growing at 51-82 degrees C was expressed in Escherichia coli JM109. The enzyme had a half-life of 10 min at 89.2 degrees C. Purification of the enzyme and its characterization showed that the enzyme was identical with the native one. Its primary structure of 529 residues with a molecular weight of 61,469 deduced from the gene was 40-42% identical to the sequences of less thermostable oligo-1,6-glucosidases from Bacillus cereus ATCC 7064, Bacillus coagulans ATCC 7050, and Bacillus thermoglucosidasius KP1006. Sequence analysis showed that the B. flavocaldarius enzyme shared 14 proline residues at the same positions as in the three other enzymes, and that the B. flavocaldarius enzyme had 22 of 33 additional proline residues (cf. 1/5, 5/10, and 9/18 in the respective counterparts) in three long polypeptides constituting the active-site cleft, which connected the third, fourth, and eighth beta-strands to the corresponding third, fourth, and eighth alpha-helices in the (beta/alpha)8-barrel.
编码嗜热黄芽孢杆菌KP1228（FERM-P9542）在51-82摄氏度生长时的细胞寡聚-1,6-葡萄糖苷酶（糊精6-α-D-葡聚糖水解酶，EC 3.2.1.10）的基因在大肠杆菌JM109中表达。该酶在89.2摄氏度下的半衰期为10分钟。对该酶的纯化及其特性表征表明，该酶与与与天然酶相同。从该基因推导出来的由529个残基组成、分子量为61469的一级结构，与蜡样芽孢杆菌ATCC 7064、凝结芽孢杆菌ATCC 7050和嗜热葡糖苷芽孢杆菌KP1006中热稳定性较低的寡聚-1,6-葡萄糖苷酶的序列有40%-42%的同源性。序列分析表明，嗜热黄芽孢杆菌的这种酶与其他三种酶在相同位置共有14个脯氨酸残基，并且在构成活性位点裂隙的三条长多肽中，嗜热黄芽孢杆菌的这种酶在另外33个脯氨酸残基中有22个（分别与相应对应物中的1/5、5/10和9/18相比），这些多肽将（β/α）8桶中的第三条、第四条和第八条β链与相应的第三条、第四条和第八条α螺旋连接起来。
