The search for rennet substitutes such as microbial rennet has increased fold due to increase in the demand for cheese products. Microbial rennet covers about one-third of the cheese consumption worldwide. Hence it is important to develop commercially viable and cost efficient method for purification of rennet from microbial sources. Hence the present work was focused on the production and purification of microbial rennet from Aspergillus candidus. The rennet was purified using a two step purification process involving solvent precipitation and chromatographic separation. The purity of the milk clotting enzyme was increased by 7.43 fold by solvent precipitation using equal mixture of 150% (v/v) ethanol-acetone. Then the enzyme was further purified using Diethylaminoethyl (DEAE) cellulose chromatography and 10.45 fold increase in enzyme activity was obtained after purification. The temperature of 35°C and substrate concentration of 0.25 mg/ml were found as optimum for maximum enzyme activity. The kinetics of the purified enzyme was studied and the Michaelis-Menten parameters such as rate constant (Km) and the maximum reaction rate (Vmax) were found as 0.059 mg/ml and 8.59 x 10-3 mmol/ml/sec respectively.

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