Европейский журнал экспериментальной биологии Открытый доступ

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Semi-purification and kinetic study of microfungal rennet biosynthesized by local isolate of Rhizomucor nainitalensis using solid-state fermentation system: Concentration methods and determinant factors in clotting activity

Khademi Farshada, Abachi Soheilab and Malekzadeh Fereydoon A.b

Our laboratory, previously, isolated and preserved at hermophilic Zygomycete, an indigenous Rhizomucor nainitalensis, demonstrating a high level of milk clotting activity for its external aspartic proteases. Following the determination of the optimum cultural conditions for the production of the microfungalrennet in solid state fermentation (SSF) system, enzymatic properties of crude rennet and the determinant factors in clotting activity were examined. The extracted rennet revealed the highest milk coagulating activity (1920 SU/ml) when was cultured on wheat bran at optimized fermentation condition of 40°C, 50% humidity, and inoculated with 103 spore/g wheat bran for 110 hours. The pH of 4.6 was found to be the optimum pH for clotting activity. Milk clotting activity increased significantly as calcium concentration in the enzyme environment was raised from 0.001 M to 0.05 M and the reaction temperature from 25°C to 65°C. It was also found that, at 75°C and the optimum pH, the enzyme becomes inactive in a period of 30 minutes. At pH of 4 and 5, the rennet maintains 100% of its activity level for 48 hours at 65°C. In addition, like the commercial rennet, the produce drennet reaches its highest level of proteolytic activity at pH of 4. As a disadvantage, the proteolytic activity of crude rennet produced in our experiment was considerably stronger than the commercial rennet. Amongst physical and chemical methods of enzyme concentration, precipitation method with 40% and 90% saturated ammonium sulfate yielded the highest level of clotting activity.