Isolation and Activity Determination of Enzyme Phosphatase Secreted by Aspergillus niger

Abstract

The use of enzymes on industrial scale saves a lot of energy and avoids pollution, thus holding a promise for green and economically sustainable alternative strategies in industrial transformations. Generally, the fungi Aspergillus niger secretes enzymes which can be used in different industries. Thus, coming up with these enzymes in large amounts will definitely result in reduced costs encountered in importing them for industrial use. This study focussed on isolation and activity determination of an enzyme phosphatase secreted by Aspergillus niger. This enzyme can be of great importance in molecular biology industries, particularly for recombinant DNA technology. For this study, pure cultures of Aspergillus niger were used. Aspergillus niger was resuscitated on potato dextrose agar and then subcultured in Adam’s medium, a medium specific for the production of phosphatase. Cells were centrifuged and the filtrate was collected whilst the residue was discarded. The filtrate was expected to contain the crude enzyme phosphatase since Aspergillus niger secretes the extracellular enzyme into the medium. Disodium phenyl phosphate was used as a substrate for the determination of the phosphatase activity. The enzyme activity was determined spectrophotometrically by reading absorbance of phenol formed in the presence of enzyme and the substrate. The concentration of phenol liberated was then used to calculate the enzyme activity expressed in King Armstrong Units (KAU). Further work on enzyme activity determination was done by varying enzyme and substrate concentrations. Results showed that the isolated alkaline phosphatase had activity of 4.0 KAU and 4.5 KAU at 25 ºC and 37 ºC respectively. Acidic phosphatase had activity of 5 KAU and 7 KAU at 25 ºC and 37 ºC respectively. Rate of activity increased upon increasing enzyme concentration and substrate. Thus, Aspergillus niger produces the enzyme phosphatase, however, there is need to induce the production of these enzymes for industrial use.