Sequential Optimization and Purification of α-Galactosidase from Actinoplanes utahensis: A way for Enhanced Production and Industrial Prospects

Abstract

Abstract α-Galactosidase is an important exoglycosidase belonging to the hydrolase class of enzyme, which has therapeutic and industrial potential. It plays a crucial role in hydrolyzing α-1,6 linked terminal galacto-oligosaccharide residues such as melibiose, raffinose, and branched polysaccharides such as Galacto-glucomannans and Galactomannans. In this study Actinoplanes utahensis was explored for α-Galactosidase production, yield improvement and activity enhancement by purification. Initially nine media components were screened by using the Plackett-Burman design (PBD). Among these components, sucrose, soya bean flour, and sodium glutamate were identified as best supporting nutrients for highest enzyme secretion by A. utahensis. Later, the Central Composite Design (CCD) was implemented to fine-tune the optimization of these components. Based on sequential statistical optimization methodologies, a significant, 3.64-fold increase in α-galactosidase production, from 16 to 58.37 U/mL was achieved. The enzyme was purified by ultrafiltration-I followed by multimode chromatography and ultrafiltration-II. The purity of the enzyme was confirmed by Sodium Dodecyl Sulphate –Polyacrylamide Agarose Gel Electrophoresis (SDS-PAGE) which revealed a single distinctive band with a molecular weight of approximately 72kDa. Additionally, it was determined that this process resulted in a 2.03-fold increase in purity. The purified α-galactosidase showed an activity of 2304 U/mL with a specific activity of 288 U/mg. This study demonstrates the feasibility of scalable α-galactosidase production, which has various industrial applications.