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

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 proteinhomogeneity (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 conditionsemployed 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 isolatedprotease activity.Key words: Mucor pusillus, protease, purification, enzymatic performance, electrophoresis, milk clotting,rennet

Isolation and identification of bacterial strain I33M producing milk-clotting enzyme: Optimization of culture parameters using response surface

A strain I33M which produces a milk-clotting enzyme was screened from Algerian soil near a dairy factory. This strain was identified as Bacillus mojavensis based on morphology and internal transcription spacer sequence. Sequencing analysis of 16S rDNA gene showed 100% identity of the tested strain with the B. mojavensis in the database. Phylogenetic analysis of this strain showed that it was most closely related to Bacillus subtilis strain. The optimum levels of these significant parameters to obtain the highest milk clotting activity and the lowest proteolytic activity were determined employing the response surface methodology (RSM), which revealed these as follows: wheat bran 7%, casein 0.094%, temperature 39°C, agitation size (rpm) 150. Among the various variables screened, agitation and temperature were most significant in submerged fermentation (SmF). The optimal value of milk clotting activity (MCA) is esteemed at 2.40. Key words: Milk clotting protease, Bacillus, response surface methodology, sequencing analysis

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

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 activit

Extracellular protease from mucor pusillus : purfication and characterization

Extracellular protease from Mucor pusillus was purified 18-fold with 7.56% recovery by ion-exchange chromatography and gel filtration. The enzyme was found to be monomeric in nature, having a molecular mass of 49 kDa. The enzyme acted optimally at 50°C and was stable in the temperature range 30–50°C. It was completely inactivated by heating for 30 min at 65°C. The optimum of activity for the purified extract was observed at milk CaCl2 concentration of 0.02 m and at milk pH of 5. These properties, except for temperature, were similar to those of renne