Purification and Characterization of a Milk Clotting Protease fromMucor bacilliformis

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.

Aspartic proteinases from Mucor spp. in cheese manufacturing

“…Fernandez-Lahore et al (1998) recovered the milk-clotting enzyme from M. bacilliformis from SSF crude extracts with 46% yield by employing ethanol (or acetone) precipitation at optimized pH values (e.g., ranging from 4.0 to 5.0). Areces et al (1992) further purified such aspartic proteinase by employing ion exchange chromatography. Combination of various methods is a common strategy: Diaz and Ruiz Herrera (1987) purified an aspartic proteinase from M. rouxii submerged cultures by a method which involved salt and acid precipitation, anion exchange chromatography, and size-exclusion chromatography.…”

“…Some of the producer strains were identified as Mucor racemosus, Mucor fragilis, Mucor mucedo, Mucor spinosus, Mucor rouxii, Mucor varians, Mucor bacilliformis, and Rhizomucor hiemalis. Further and more detailed studies focused on the biosynthesis of aspartic proteinases by M. bacilliformis (Areces et al 1992), M. varians var Pispek (Bernardinelli et al 1983), M. mucedo (Handel and Fraile 1984), and Mucor renninus (Belyauskaite et al 1980). Tubesha and Al-Delaimy (2003) studied 20 Mucor spp.…”

Milk-clotting protease production by Nocardiopsis sp. in an inexpensive medium

“…Other authors are in agreement with these results. Hashem (1999) and Areces et al (1992), working respectively with Penicillium oxalicum and Mucor baciliformis, noted that glucose was not the most favourable carbon source for milk-clotting enzyme production. Figure 2 shows the milk-clotting activity curves of Nocardiopsis sp.…”

“…The milk-clotting activities determined in M3 and M4 were low (11 and 2.55 U/ml, respectively), suggesting that induction effects of the soybean flour may be an important factor. The synthesis and secretion of protease are induced by peptides or other proteinaceous substrates, such as soybean flour (Porto et al 1996) and wheat bran (Areces et al 1992). Depending on the peptide nature and level, protease synthesis and secretion may be induced or repressed (Porto et al 1996).…”

“…For a long time calf rennet has been the traditional source of milk-clotting enzymes for cheese manufacturing, but decreasing worldwide rennet supplies, accompanied by ever increasing cheese production and consumption, made necessary the utilization of rennet substitutes (Areces et al 1992).…”