Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan.
Extremophiles. 2012 May;16(3):395-403. doi: 10.1007/s00792-012-0438-z. Epub 2012 Mar 6.
The smallest and enzymatically active molecule, TetApuQ818, was localized within the C-terminal Q818 amino acid residue after serial C-terminal truncation analysis of the recombinant amylopullulanase molecule (TetApuM955) from Thermoanaerobacter pseudoethanolicus. Kinetic analyses indicated that the overall catalytic efficiency, k (cat)/K (m), of TetApuQ818 was 8-32% decreased for the pullulan and the soluble starch substrate, respectively. Changes to the substrate affinity, K (m), and the turnover rate, k (cat), were decreased significantly in both enzymatic activities of TetApuQ818. TetApuQ818 exhibited less thermostability than TetApuM955 when the temperature was raised above 85°C, but it had similar substrate-binding ability and hydrolysis products toward various substrates as TetApuM955 did. Both enzymes showed similar spectroscopies of fluorescence and circular dichroism, suggesting the active folding conformation was maintained after this C-terminal Q818 deletion. This study suggested that the binding ability of insoluble starch by TetApuM955 did not rely on the putative C-terminal carbohydrate binding module family 20 (CBM20) and two FnIII regions of TetApu, though the integrity of the AamyC module of TetApuQ818 was required for the enzyme activity.
经串联 C 端截断分析，来自热厌氧菌的重组支链淀粉 pullulanase 分子（TetApuM955）中，最小且具有酶活性的分子 TetApuQ818 定位于 C 端 Q818 氨基酸残基内。动力学分析表明，TetApuQ818 对 pullulan 和可溶性淀粉底物的总体催化效率 k（cat）/K（m）分别降低了 8-32%。两种酶活性中，TetApuQ818 的底物亲和力 K（m）和周转率 k（cat）均显著降低。当温度升高到 85°C 以上时，TetApuQ818 的热稳定性低于 TetApuM955，但与 TetApuM955 一样，它对各种底物具有相似的底物结合能力和水解产物。两种酶的荧光和圆二色性光谱相似，表明在 C 端 Q818 缺失后，活性折叠构象得以保持。本研究表明，尽管 TetApuQ818 的 AamyC 模块的完整性对于酶活性是必需的，但 TetApuM955 对不溶性淀粉的结合能力并不依赖于假定的 C 端碳水化合物结合模块家族 20（CBM20）和两个 FnIII 区。
