Purification and Characterization of Proteolytic Enzymes from a Fungi Isolated from Poultry Waste

Akinriade, Oluwatosin Mary (2021)

xviii,85 P

Thesis

Thesis

This study screened, isolated and identified a proteolytic fungus from poultry waste of the Obafemi Awolowo University farm, Ile-Ife, Osun State, Nigeria and subsequently characterised the best protease producing isolate. Protease produced was purified to homogenity and characterised. These were with a view to producing proteolytic enzyme for industrial applications. Poultry waste samples were collected from two points (Latitude N 7033’7.914’ and Longitude E 4033’6.972) at Obafemi Awolowo University Poultry Farm, Ile-Ife. The samples were dissolved in sterile distilled water, serially diluted on Sabouraud extract agar for isolation of fungi. The influence of various parameters was studied to optimize protease production such as incubation time, inoculum size, carbon and nitrogen sources, pH and temperature. The isolated fungi from the poultry waste sample were purified, identified by morphological techniques and screened for protease production. Protease was produced by the isolated fungus using submerged fermentation, concentrated by lyophilisation and purified to homogeneity using gel filtration chromatography. The molecular weight of the enzyme was determined by sodium dodecyl sulphate-polyacrylamide gel-electrophoresis (SDS-PAGE). The biochemical properties of the purified enzyme such as kinetic parameters, effects of inhibitors, pH, salts, solvents and surfactants were investigated using standard techniques. The result obtained showed that out of the six fungal strains which had proteolytic activity, Aspergillus flavus was the best protease producing strain. The strain produced protease at pH 10, inoculum size of 16 mm and temperature 35 oC, respectively. The maximum activity was obtained in a medium containing casein, urea, yeast extract for nitrogen sources and glucose as carbon source. After gel filtration chromatography using Sephacryl S-200, the molecular weight of the purified protease obtained from SDS-PAGE was 33.35 kDa. The kinetic parameters of Aspergillus flavus protease with casein as substrate are 0.418±0.05 mg/ml, 0.05214±0.1597 µmoles/ml/min and 1.53 s-1 for km, Vmax and kcat, respectively. The optimum pH was 10 and remained stable after 90 min incubation. Optimum temperature was obtained at 50oC and was stable after incubating for 120 min with the activation energy (Ea) of 20.3 kJ/mol. The purified enzyme was active only at 1 mM Na+, K+, Mn2+ and Mg2+ while Ca2+ and Cu2+ salts activated the enzyme at higher concentration. Phenylmethylsulfonide fluoride (PMSF) greatly inhibited the activity of protease at 1 mM, indicating that purified enzyme is a serine-protease. The purified enzyme showed excellent activity in various surfactants and oxidizing agent at 1% and Tween-20 at 5%. The study concluded that a novel strain of Aspergillus flavus isolated from poultry waste was able to produce a serine-protease which had unique biochemical properties such as thermal and surfactant stability making this enzyme of a great importance in several industrial and biotechnological applications.