Efficient Utilization of Dairy Industry Waste for Hyper-Production and Characterization of a Novel Cysteine Protease

Dairy industry waste (fresh cheese whey) has been utilized as a sole substrate for the production of extracellular protease by a locally isolated strain of Rhizopus oryzae. In pure whey medium only mineral salts were added as (g/L): MnSO4.H2O (0.01%), FeSO4.H2O (0.01%), CaCl2 (0.05%) at pH 6. Effects of shaking speed, pH, temperature and incubation time for protease production were studied in submerged fermentation process. Maximum production of extracellular alkaline protease was achieved after 168 h at 35C. To further improve the production from this strain mutagenesis was performed. Strain improvement using EMS culminated in a mutant ROAC5 with a 2.2 fold increased yield and low volumetric substrate consumption. Partial characterization was performed to expose the enzyme properties, obtained from parent and mutant strains. ANOVA confirmed that the enzyme produced from mutant strain was novel, thermo-tolerant and active at pH 10, resulting in 3.85 fold elevated enzyme activity (2538.9 PU/ml), under optimum fermentation conditions. This is the first study to report a novel cysteine alkaline protease produced by Rhizopus oryzae.