English abstract
Proteases are protein hydrolysing enzymes. More than 60% of the industrial enzymes are proteases with a wide range of applications. Thermostable bacteria are capable of producing thermostable proteases that can resist denaturation due to extreme temperature, pH and chemicals. The guts from Sardinella longiceps were processed and used as substrate for low cost production of protease enzyme. Fish waste contained 61.6 % proteins, 21.8% lipids, 8.5% carbohydrates and trace elements such as K, Na+, Ca2+, Mg2+, Fe2+, Zn*, P3- and salts PO4, C12, SO42- and NO3. Soil samples were collected from Nakhi hot spring in Sultanate of Oman to isolate protease producing bacteria. From 101 isolates, one bacteria that produced the highest protease activity (51.8 U/ml) was identified as Bacillus licheniformis. The protease enzyme produced under optimized conditions was purified and characterized. Optimization of protease. production conditions was carried out using Placket Burman (PB) design and Response Surface Methodology (RSM). Positive factors for protease fermentation were studied using PB design with 13 runs using combinations of five factors. The factor ranges were temperature (40-80°C), pH (7-12), time (24-72 h), substrate concentration (0.5-2%), and inoculum size (1-5%). Based on PB results, factors and levels for RSM design were selected. The factors and levels were temperature (40 50°C), time (65-75 h), substrate concentration (1.5-3%), and inoculum size (4-5%) and the design generated 31 runs with different combinations. Model was significant (R2 – 82.92%) and production under optimized conditions produced an increase of 1.5-fold in activity resulting in 73.52 U/ml. Purified enzyme produced a final yield of 1.70% with a 6.46-fold purification. Molecular weight of the enzyme was determined by SDS-PAGE as 60 and 65 KDa with two corresponding bands. The purified protease was characterized as alkaline and thermo tolerant protease with its optimum pH at 9 and temperature at 50°C. Protease activity was enhanced by Zn++ and Nat and was inhibited by other metal ions. Protease was stable against urea, B-mercaptoethanol, sodium dodecyl sulphate (SDS) and tween 80. Protease enzyme was compatible with four commercial detergents such as Arial, Bahar, Bonux and Tide and the best substrate for maximum activity was tryptone soya. The protease had a maximum velocity of 88.57 U/ml and Km equivalent to 0.4384 mg/ml. Thus, the fish waste could be used as a cheaper substrate for protease production and the Nakhl hot spring is a good source for isolating bacteria with biotechnological potentials..
