Investigating the effect of SUMO fusion on solubility and stability of amylase-catalytic domain from Pyrococcus abyssi.

Alpha amylase belonging to starch hydrolyzing enzymes has significant contributions to different industrial processes. The enzyme production through recombinant DNA technology faces certain challenges related to their expression, solubility and purification, which can be overcome through fusion tags. This study explored the influence of SUMO, a protein tag reported to enhance the solubility and stability of target proteins when fused to the N-terminal of the catalytic domain of amylase from Pyrococcus abyssi (PaAD). The insoluble expression of PaAD in E. coli was overcome when the enzyme was expressed in a fusion state (S-PaAD) and culture was cultivated at 18 °C. Moreover, the activity of S-PaAD increased by 1.5-fold as compared to that of PaAD. The ligand binding and enzyme activity assays against different substrates demonstrated that it was more active against 1 % glycogen and amylopectin. The analysis of the hydrolysates through HPLC demonstrated that the enzyme activity is mainly amylolytic, producing longer oligosaccharides as the major end product. The secondary structure analyses by temperature ramping in CD spectroscopy and MD simulation demonstrated the enzymes in the free, as well as fusion state, were stable at 90 °C. The soluble production, thermostability and broad substrate specificity make this enzyme a promising choice for various foods, feed, textiles, detergents, pharmaceuticals, and many industrial applications.