Revealing the role of the X25 domains through the characterization of truncated variants of amylopullulanase enzyme from Thermoanaerobacter brockii brockii.

Autor: Kayrav A; 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., Mumcu H; 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., Durmus N; 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., Karaguler NG; 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. Electronic address: karaguler@itu.edu.tr.
Jazyk: angličtina
Zdroj: International journal of biological macromolecules [Int J Biol Macromol] 2024 Jun; Vol. 270 (Pt 2), pp. 132404. Date of Electronic Publication: 2024 May 14.
DOI: 10.1016/j.ijbiomac.2024.132404
Abstrakt: 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 m and higher k cat /K m 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 m and lower k cat /K m 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.
Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Nevin Gul Karaguler reports financial support was provided by the Istanbul Technical University Scientific Research Projects Coordination Unit. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2024 Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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