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Purification and characterization of a milk clotting protease from Mucor bacilliformis
L B Areces1, M B Bonino, M A Parry
1Instituto de Quimica y Fisicoquimica Biológicas, UBA-CONICET, Argentina.
Abstract:
An acid protease having milk clotting activity has been isolated from Mucor bacilliformis cultures. The enzyme was basically purified by ionic exchange chromatography. An average yield of 29 mg purified product was obtained from 100 mL crude extract. As purity criteria, SDS-PAGE, reverse-phase HPLC, and N-terminal analysis were performed. The protease is a protein composed of a single polypeptide chain with glycine at the N-terminus. The mol wt is approx 32,000, and its amino acid composition is very similar to those of other fungal proteases. As expected, its clotting activity was drastically inhibited by pepstatin A action. On the other hand, its instability against heat treatment and its clotting/proteolytic activity ratio indicate that it may be considered as a potential substitute for bovine chymosin.
Insights
Researchers isolated an acid protease from Mucor bacilliformis with milk-clotting properties. This fungal protease shows potential as a substitute for bovine chymosin in various applications.
Area of Science:
- Enzymology
- Microbiology
- Food Science
Background:
- Milk clotting enzymes are crucial in dairy processing.
- Bovine chymosin, the traditional enzyme, faces supply challenges.
- Exploring alternative microbial proteases is essential for sustainable dairy production.
Purpose of the Study:
- To isolate and characterize an acid protease with milk-clotting activity from Mucor bacilliformis.
- To evaluate its potential as a substitute for bovine chymosin.
Main Methods:
- Isolation and basic purification of the acid protease using ion-exchange chromatography.
- Purity assessment via SDS-PAGE, reverse-phase HPLC, and N-terminal analysis.
- Characterization of enzyme properties, including molecular weight and amino acid composition.
Main Results:
- An acid protease was successfully isolated and purified from Mucor bacilliformis cultures.
- The enzyme is a single polypeptide chain with a molecular weight of approximately 32,000 Da and glycine at the N-terminus.
- Clotting activity was inhibited by pepstatin A, and the enzyme exhibited instability to heat treatment.
Conclusions:
- The characterized acid protease from Mucor bacilliformis possesses significant milk-clotting activity.
- Its properties suggest it could be a viable alternative to bovine chymosin in dairy applications.
- Further research into its stability and activity ratio is warranted for commercial viability.