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

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.

Cloning and characterization of thermostable amylopullulanase TbbApu and its C-terminal truncated variants with enhanced activity in organic solvents.

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 this study, we focused to elucidate the complete sequence of the apu gene and the role of C-term domains on biochemical properties and enzyme activity of Thermoanaerobacter brockii brockii amylopullulanase (TbbApu). After the gene sequence was defined, C- term truncated variants were constructed. The most suitable host organism and expression vector were determined as E. coli BL21(DE3) and pET-28a(+) depending on the highest yield/biomass ratio for recombinant production of all constructs. It was seen that the expression yield increased approximately threefold in the case of the SH3 region truncation. In the biochemical characterization, TbbApu and its truncated variants exhibited maximum activity at 70 °C and 75 °C for pullulan and starch hydrolysis respectively, and the optimum pH of TbbApu were 6.5 and 6 for truncated variants. Moreover, hydrolysis activities of all recombinant enzymes were enhanced by Mn2+, Co2+ and Cu2+, detergents, and almost all organic solvents; except butanol, DMF and DMSO. All recombinant amylopullulanases remained 80% stable up to 80 °C in the wide range of pH and also retained > 85% stability in the presence of defined volatile organic solvents. No significant difference was observed between the raw starch adsorption capacity and the specific activity of the three variants. These results indicated that the C-terminal regions of TbbApu are non-essential for the enzyme activity, stability and substrate binding capacity; furthermore, hexane and acetone organic solvents enhanced both pullulanase and α-amylase activity of these enzymes, interestingly. With these features, TbbApu and its truncated variants are distinguished from other thermophilic amylopullulanases and also make them promising candidates for industrial use.