PURIFICATION AND CHARACTERIZATION OF PHOSPHATASES FROM ASPERGILLUS FUMIGATUS FOR APPLICATIONS IN INDUSTRIAL USES

摘要

The present study aims on the successful isolation, production, purification and characterization of acid phosphatase, alkaline phosphatase and phytase enzymes from Aspergillus fumigatus in terms of its industrial applications. Since soil is a reservoir for the isolation of such fungi, soil microbes were screened for phosphatases and a strain of Aspergillus fumigatus was isolated. For this study, 25 protein rich soil samples were collected from Kengeri gardens of Bangalore, Karnataka, India. . A typical fungal colony isolated after serial dilution, identified as A. fumigatus was used in this study. On molecular identification, the strains identified on the basis of morphological and physiological features were confirmed to belong to A. fumigatus. Their ITS1-ITS4 sequences were up to 99% homologous to reference strains of A. fumigatus from the GenBank. After purification of the culture, culture conditions were standardized. Acid phosphatase, alkaline phosphatase and phytase Enzyme activity assays and protein estimation activity by Lowry’s method was done for acid phosphatase, alkaline phosphatase and phytase of A. fumigatus. The sample was purified by ammonium salt precipitation, dialysis and ion exchange chromatography. The enzymes assay activity was determined for crude sample and after every stage of purification i.e. dialysis and ion-exchange chromatography. SDS PAGE analysis showed that the acid phosphatase corresponded to 50 KDa molecular mass. While the molecular mass of the fungal phytase was 47 KDa, which of alkaline phosphatase enzyme was 60 KDa. Alkaline phosphatase exhibited specific activity of 0.23 U/mg, pH and temperature optima for the enzyme activities were 8.0 and 40°C for alkaline phosphatase, Acid phosphatase exhibited specific activity of 0.14 U/mg, pH and temperature optima for the enzyme activities were 7.0 and 50°C for acid phosphatase and Phytase exhibited specific activity of 0.113 U/mg, pH and temperature optima for the enzyme activities were 6.0 and 50°C for phytase. Due to its pH profile and optimum, it could be an interesting candidate for biotechnological applications. Phytase is a promising candidate for applications in the feed industry to enhance the quality for poultry and piggery by supplementing it in their diets. Alkaline phosphatase has become an important tool in molecular cloning and DNA sequencing. It also used as an important part of diagnostic kits component of different ELISA base kits. These enzymes have considerable potential for many industrial and commercial applications.