Istanbul Technical University, Faculty of Science and Letters, Department of Molecular Biology and Genetics, 34469 Istanbul, Türkiye; Istanbul Technical University, Dr. Orhan Öcalgiray Molecular Biology-Biotechnology and Genetics Research Center, Istanbul, Türkiye.
Istanbul Technical University, Faculty of Science and Letters, Department of Molecular Biology and Genetics, 34469 Istanbul, Türkiye; Istanbul Technical University, Dr. Orhan Öcalgiray Molecular Biology-Biotechnology and Genetics Research Center, Istanbul, Türkiye.
Int J Biol Macromol. 2024 Jun;270(Pt 2):132404. doi: 10.1016/j.ijbiomac.2024.132404. Epub 2024 May 14.
To understand the role of the X25 domains of the amylopullulanase enzyme from Thermoanaerobacter brockii brockii (T. brockii brockii), four truncated variants that are TbbApuΔX25-1-SH3 (S130-A1484), TbbApuΔX25-2-SH3 (T235-A1484), TbbApuΔX25-1-CBM20 (S130-P1254), and TbbApuΔX25-2-CBM20 (T235-P1254) were constructed, expressed and characterized together with the SH3 and CBM20 domain truncated variants (TbbApuΔSH3 (V1-A1484) and TbbApuΔCBM20 (V1-P1254). TbbApuΔSH3 showed improved affinity and specificity for both pullulan and soluble starch than full-length TbbApu with lower K and higher k/K values. It indicates that SH3 is a disposable domain without any effect on the activity and stability of the enzyme. However, TbbApuΔX25-1-SH3, TbbApuΔX25-2-SH3, TbbApuΔX25-1-CBM20, TbbApuΔX25-2-CBM20 (T235-P1254) and TbbApuΔCBM20 showed higher K and lower k/K values than TbbApuΔSH3 to both soluble starch and pullulan. It specifies that the X25 domains and CBM20 play an important role in both α-amylase and pullulanase activity. Also, it is revealed that while truncation of the CBM20 domain as starch binding domain (SBD) did not affect on raw starch binding ability of the enzyme, truncation of both X25 domains caused almost complete loss of the raw starch binding ability of the enzyme. All these results enlightened the function of the X25 domains that play a more crucial role than CBM20 in the enzyme's binding to raw starch and also play a crucial role in its activity.
为了理解嗜热厌氧菌（Thermoanaerobacter brockii brockii）来源的支链淀粉酶（amylopullulanase enzyme）中 X25 结构域的作用，构建了四个截断变体，即 TbbApuΔX25-1-SH3（S130-A1484）、TbbApuΔX25-2-SH3（T235-A1484）、TbbApuΔX25-1-CBM20（S130-P1254）和 TbbApuΔX25-2-CBM20（T235-P1254），并与 SH3 和 CBM20 结构域截断变体（TbbApuΔSH3（V1-A1484）和 TbbApuΔCBM20（V1-P1254）一起进行了表达和特性分析。TbbApuΔSH3 对普鲁兰和可溶性淀粉的亲和力和特异性均优于全长 TbbApu，表现出较低的 K 和较高的 k/K 值。这表明 SH3 是一个无任何作用的可抛弃结构域，不会影响酶的活性和稳定性。然而，TbbApuΔX25-1-SH3、TbbApuΔX25-2-SH3、TbbApuΔX25-1-CBM20、TbbApuΔX25-2-CBM20（T235-P1254）和 TbbApuΔCBM20 对可溶性淀粉和普鲁兰的 K 值更高，k/K 值更低。这表明 X25 结构域和 CBM20 在α-淀粉酶和支链淀粉酶活性中均发挥着重要作用。此外，研究结果还表明，尽管 CBM20 结构域的截断作为淀粉结合结构域（SBD）并不影响酶对生淀粉的结合能力，但两个 X25 结构域的截断几乎完全丧失了酶对生淀粉的结合能力。所有这些结果都阐明了 X25 结构域的功能，它们在酶与生淀粉的结合中比 CBM20 发挥着更关键的作用，并且在其活性中也发挥着关键作用。
