Isolation, physico-chemical characterization and biological activity of α-amylase from the culture liquid of the fungus Aspergillus oryzae for using in the enzyme-linked immunosorbent assay

Abstract

The article describes the development of an effective method for purifying the α-amylase enzyme from the culture liquid (CL) of the fungus Aspergillus oryzae, by using an affinity sorbent, and the development of optimal conditions for sorption and desorption of proteolytic enzymes from CL on protein-impregnated Sorsilene. The purification of α-amylase from the CL of the fungus Aspergillus oryzae was studied by salting, ion exchange, and gel chromatography with and without removal of proteinases. It was established that in the process of isolating α-amylase from the CL of the fungus Aspergillus oryzae, the preliminary removal of proteinase by its biospecific sorption contributes to an increase in the efficiency of enzyme purification. Some physicochemical characteristics and biological activity of the obtained highly purified enzyme have been investigated. It has been shown that the preliminary removal of proteinases contained in CL on biospecific sorbents contributes to an increase in the degree of purification of α-amylase. An ELISA was developed to detect the bacterial antigen, a plant virus, and a pathogenic fungus, in particular, diphtheria toxin, the tobacco mosaic virus TS-TMV, and the protein antigen of the fungus P. varioti, using α-amylase as an enzyme label. The developed method has a number of advantages in comparison with the known ELISA methods, where horseradish peroxidase and phospholipase A2 enzymes were used.