Aycan Kayrav , Hande Mumcu, Naciye Durmus, Nevin Gul Karaguler
DOI: 10.1016/j.ijbiomac.2024.132404
The article was published in the International Journal of Biological Macromolecules (Q1) as a part of Aycan Kayrav`s MSc thesis.
Bifunctional debranching-enzyme amylopullulanases belong to the glycoside hydrolases (GHs) family and catalyze both the hydrolysis of α-1,4 and α-1,6 glycosidic bonds in starch, pullulan, amylopectin and glycogen polysaccharides. Among these, especially thermostable ones are essential in starch processing applications. In our previous study, the amylopullulanase enzyme (TbbApu) from the thermophilic, anaerobic bacterium Thermoanaerobacter brockii brockii (T. brockii brockii) and its two truncated variants which are TbbApuΔSH3 and TbbApuΔCBM20 was recombinantly produced and
characterized (Mumcu et al. 2023). To understand the role of the X25 domains of the, 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.
1.) . Mumcu, H., Kayrav, A., Isleyen, N.D., Karaguler, N.G. (2023) Cloning and characterization of thermostable amylopullulanase TbbApu and its C-terminal truncated variants with enhanced activity in organic solvents, Enzym. Microb. Technol. 164, 110176 (It was published as a part of Hande Mumcu`s MSc thesis).