Extracellular Proteases of Mucor pusillus

Area of Science:

• Microbiology
• Enzymology
• Biochemistry

Background:

• Mucor pusillus is a fungus known for producing enzymes with milk-clotting and protease activities.
• Previous research indicated the presence of a single major acid protease in M. pusillus.
• Optimizing media composition is key to maximizing desired enzyme production.

Purpose of the Study:

• To investigate the milk-clotting and protease activities of Mucor pusillus grown in various media.
• To characterize the proteases produced by M. pusillus, particularly focusing on rennin-like activity.
• To develop a method for separating the rennin-like enzyme from other proteases.

Main Methods:

• Cultivation of Mucor pusillus in media supplemented with corn steep liquor and glucose.
• Assaying extracellular milk-clotting and protease activities.
• Ion-exchange chromatography and polyacrylamide gel electrophoresis for protease separation and analysis.
• Ammonium sulfate fractionation for enzyme purification.

Main Results:

• The highest ratio of milk-clotting activity to protease activity was observed in a medium containing 3% corn steep liquor and 1% glucose.
• Two major acid proteases were identified, contrary to previous findings of a single protease.
• One protease exhibited significant rennin-like activity, while the other did not.
• Electrophoresis confirmed the presence of two distinct protease activity bands.
• Ammonium sulfate fractionation effectively removed a substantial portion of the non-specific protease activity.

Conclusions:

• Mucor pusillus produces at least two distinct acid proteases, one with rennin-like activity.
• The identified rennin-like protease is potentially valuable for dairy applications.
• A purification strategy involving ammonium sulfate fractionation and ion-exchange chromatography can separate the desired rennin-like enzyme.