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

Aycan Kayrav, Hande Mumcu, Naciye Durmus, Nevin Gul Karaguler*

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Özet

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 Km and higher kcat/Km 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 Km and lower kcat/Km 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.

Orijinal dilİngilizce
Makale numarası132404
DergiInternational Journal of Biological Macromolecules
Hacim270
DOI'lar
Yayın durumuYayınlandı - Haz 2024

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Publisher Copyright:
© 2024 Elsevier B.V.

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