Purification and Characterization of Aspartic Protease Produced from Aspergillus oryzae DRDFS13 MN726447 under Solid-State Fermentation

Aspartic proteases (E.C.3.4.23.) are endopeptidases with molecular masses ranging between 30-45 kDa. They depend on aspartic acid residues for their catalytic activity and show maximal activity at low pH. Thus the main objective of the present study was to purify and characterize aspartic protease from locally identified fungi. The aspartic protease in the current study was obtained from A. oryzae DRDFS13 under SSF (solid-state fermentation). The crude enzyme extract was purified by size-exclusion (SEC) and ion-exchange (IEC) chromatography. The milk-clotting activity (MCA), protease activity (PA); the presence of N-glycosylation, inhibition studies and molecular weight were determined using standard methods. Optimum temperature and stability, optimum pH and stability on MCA were assessed using standard methods. The highest MCA (477.11 U/mL), specific activity (183.50 U/mg), purification fold (6.20) and yield (9.2%) were obtained from IEC fraction A(8). The molecular weight of 40 kDa was assigned for IEC A(8). The protein was glycosylated. Incubation of the IEC A(8) with pepstatin A caused a 94% inhibition on MCA. The enzyme showed maximum MCA at 60 degrees C and pH 5.0 with stability at pH 4.5-6.5 and temperature 35-45 degrees C. Furthermore, the results obtained in the present study confirmed that the aspartic protease enzyme produced from A. oryzae DRDFS13 could be used as a substitute source of rennet enzyme for cheese production. [GRAPHICS] .