Characterization of a recombinant endo-β-1,3-glucanase from Alkalihalobacillus Clausii KSM-K16 and its application in the production of curdlan oligosaccharides

Abstract

Curdlan polysaccharides are hydrolyzed to provide bioactive β-1,3-gluco-oligosaccharides, which are finding increasingly used in biomedical applications. Alkalihalobacillus clausii KSM-K16 (AC-GA) is an alkalophilic bacterium endo-β-1,3-glucanase. It has a predicted molecular weight of 30.09 kDa and is made up of 257 amino acids. It was expressed in Escherichia coli and purified to homogeneity. This study analyses the properties of AC-GA, product distribution, and structural aspects of AC-GA that may be relevant to sugar molecule production. The optimum reaction condition of the enzyme is 70 °C, pH 7.0, and its catalytic activity increased by Mn2+. Meanwhile, Zn2+ and Cu2+ inhibited AC-GA activity. It is worth mentioning that the isolated enzyme demonstrated remarkable stability at both high temperature, and neutral environments, when curdlan was utilized as the substrate, the enzyme activity reached 1008.98 U/mg. Hydrolysates of curdlan polysaccharide were β-1,3-gluco-oligosaccharides with polymerization (DP) degrees ranging from 1 to 5. This implies that the AC-GA active site might identify up to five glucose units. High concentration of AC-GA mostly creates glucotriose (DP3), whilst low concentration of AC-GA produces oligosaccharides with varying DP (primarily DP1, DP3, and DP4). Additionally, the catalytic triad (Glu134, Asp136, and Glu139) plays a decisive role in the activity of AC-GA, and structural analysis revealed that Trp129, Trp118, Trp114 and Trp 232 are involved in hydrophobic stacking interactions. This study provides a theoretical basis for the acquisition of curdlan oligosaccharides with higher application value under high-temperature hydrolysis.