Isolation, Production and Applications of Alkaline Protease From Hot Springs Bacterial Isolates

Microbial proteases contribute 60% of the total enzymes of the world market. Present study was aimed to isolate and optimize the protease production from bacterial isolates of hot springs located in Tuwa, Gujarat, India. Total 12 bacterial isolates were screened on skim milk agar medium for proteolytic activity. Based on relative enzyme activity MNK-1, MNK-2, and MNK-3 were selected for submerged fermentation (SmF) and characterized by cultural, biochemical and microscopic features. Optimization of protease production with respect to production time, carbon source, nitrogen source, inoculum volume, pH and temperature were studied. Protease production of 0.49, 0.5 and 0.49 U/mL were observed with pH 8 at 37°C in shaking condition, respectively by MNK-1, MNK-2 and MNK-3 within 48 h. Highest protease production 0.55 U/mL was observed in casein as compared to other nitrogen sources such as yeast extract, meet extract, tryptone, NaNO3 and peptone. Various carbon sources like starch, carboxyl methyl cellulose (CMC), glucose, Na-citrate and sucrose were tested for protease production among them CMC produced 0.55, 0.58 and 0.61 U/mL by MNK-1, MNK-2 and MNK-3. The protease production 0.9 U/mL was detected by inoculating 5% v/v inoculum of MNK-2. These three bacteria were able to produce protease in the pH range 6-10 and at pH 8.0 highest 0.9 U/mL protease produced by MNK-3 compared to MNK-1 and MNK-2. Proteases production were studied in range 20 to 65°C and at 45°C MNK-2 produced 1.1 U/mL protease after 48 h of incubation period. We also tested the crude enzyme for gelatin hydrolysis and silver extraction from X-ray films and as detergent additive in laundry.