Study the Optimum Conditions, Purification, and Characterization of the Acid Protease Rennet Substitute Produced from Rhizomucor Miehei

An isolate of Rhizomucor Miehei was identified, and registered with accession number PP955195.1 in NCBI, which was distinguished by its production of acid protease rennet substitute, and then compared with four commercial preparations in soft cheese-making, all this was done in a previous study. Continuation of that study, the highest enzyme proteolytic activity and productivity were achieved on rice bran using several agricultural and industrial wastes. The optimum conditions for the production of acid protease by solid-state fermentation included the hydration of rice bran with distilled water (1:2, V:W) at an initial pH of 6.0, the inoculation of 106 spores per gram of dry substrate, and an incubation temperature of 40 °C for 3 days. The enzyme was purified by ethanol 50–75%, DEAE-Cellulose ion exchange, and gel filtration on a Sephadex G-100 column, giving the final 13.59-fold purification and 32% yield. The purity of the enzyme to homogeneity was confirmed by polyacrylamide gel electrophoresis under non-denaturing conditions. When studying the characterization of the enzyme, it was found that the molecular weight of the purified enzyme as determined by gel filtration was 37.15 kD, and the isoelectric point of the enzyme was 5.2. The optimum pH for proteolytic activities on casein was 5.5, while the milk-clotting activity was pH-dependent and increased with decreasing the pH of reconstituted skim milk. The enzyme was most stable in a pH range of 4.0–6.0. The maximum proteolytic and milk-clotting activities were observed at 60 °C. In the thermal stability study, when treated with heat at 35 °C for 60 minutes, the enzyme maintained full proteolytic and milk-clotting activities.