African Journal of Biotechnology

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Purification and characterization of a milk-clotting protease from Mucor pusillus: Method comparison

A Nouani, F Moulti-Mati, S Belbraouet, MM Bellal


Crude enzymatic extract obtained from five fermentations (300 g of wheat bran) was characterized by a clotting activity of 0.34 ± 0.08 UP/ml with a strength ratio of 1/1: 200. The comparative study of the summaries from 2 purification protocols showed that it is possible to recover 6% of the initial proteins with a 44.54% activity after gel filtration (protocol I), which appeared more technically sound when compared to ion-exchange (1.80% of total proteins with a 23% performance) (protocol II). The protein
homogeneity (a single electrophoretic band) of the monomeric protease was confirmed by both methods after precipitation with 80% saturated ammonium sulphate. Moreover, the fractional precipitation technique with this salt (40 and 80%) was useless in the experimental conditions
employed and an important loss of activity was observed (28.53%) with a 3-fold purification. In another part of the study, without ammonium sulphate precipitation, the gel filtration enabled the elimination of almost 97% of the inactive proteins and improved the activity performance by 55.13%, while multiplying the specific activity of the coagulant by a factor of 20.88 against a 6.75-fold purification with ionexchange and the appearance of a more or less 20 kDa peptide after electrophoresis. The proteolytic activity of the purified extracts had a similar appearance to a more pronounced kinetic when compared with the reference rennet. The purification protocols did not seem to have an impact on the isolated
protease activity.

Key words: Mucor pusillus, protease, purification, enzymatic performance, electrophoresis, milk clotting,

AJOL African Journals Online