Purification and characterization of a milk clotting protease from Mucor bacilliformis

• Published in: Applied biochemistry and biotechnology 1992-12, Vol.37 (3), p.283-294
• Main Authors: ARECES, L. B, DE JIMENEZ BONINO, M. B, PARRY, M. A. A, FRAILE, E. R, FERNANDEZ, H. M, CASCONE, O
• Format: Article
• Language: English
• Subjects: Amino Acids - analysis; Animals; Aspartic Acid Endopeptidases - isolation & purification; Aspartic Acid Endopeptidases - metabolism; Biological and medical sciences; Biotechnology; Caseins - metabolism; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; cuajo; Culture Media; Electrophoresis, Polyacrylamide Gel; Enzyme engineering; Fundamental and applied biological sciences. Psychology; Hydrogen-Ion Concentration; Improved methods for extraction and purification of enzymes; Kinetics; Methods. Procedures. Technologies; microorganisme; microorganismos; microorganisms; Milk - metabolism; Molecular Weight; mucor; Mucor - enzymology; presure; proteolisis; proteolyse; proteolysis; purificacion; purification; rennet
• ISSN: 0273-2289; 1559-0291
• DOI: 10.1007/BF02788880

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.