Production Physiology and Biochemical properties of Crude Thermophile Protease by Mild - Halophilic Bacillus thuringiensis aizawai HD 865

Abstract

Objective: The current study aims to enrich the research library of Bacillus thuringensis, the most famous bacteria as a biopesticide, with a product that may enhance its ability as a biopesticide as well as its many other applications, namely the heat-tolerant alkaline protease. Methods: The strain Bacillus thuringiensis aizawai HD 865, obtained from HD Culture Collection was grown on a liquid growth medium of nutrient broth and yeast extract, and colonies were counted on agar plates. The zygote is then transferred to a skim milk environment to estimate the enzymatic activity under the different growth conditions of the growth environment (sodium chloride concentration, pH, aeration, fermentation duration), then testing the optimal conditions for enzymatic activity in the enzymatic reaction mixture (sodium chloride concentration, salinity resistance, optimum temperature, thermal stability, optimum pH and stability). Then analyze the data statistically. Results: Optimum alkaline protease medium production conditions were, 2 % NaCl, so Bacillus thuringiensis aizawai HD865 a mild - halophilic microorganism, aeration level 80%, pH 7 and 8 days of fermentation period. On the other hand, crude alkaline protease properties were found that, the optimum temperature for its activity was relatively high (70ºC), while heat-stabilities after 5 min of heating crude enzyme in boiling water bath then incubated for 15min on 30, 40, 50, 60, 70ºC, were 184.23, 178.07, 164.29, 165.51 and 121.46 Units, respectively; whereas the optimum pH was at pH 7.6 (151.76 Units) with tris buffer then at pH 9.6 with glycine - NaOH buffer; While pH stability was at pH 7.6 with tris buffer then at pH 8.4 with glycine - NaOH buffer. Thus, activities have decreased at different concentrations (0, 1, 2, 3, 4, 5 %) of sodium chloride in reaction mixture with enzyme activities (151.25, 137.35, 135.12, 135, 135.89, 114.38 Units) respectively, also decreased when rinse crude enzyme for 5 min in NaCl (0, 1, 2, 3, 4, 5 %), with (151.04, 175.57, 168.69, 154.61, 155.39 and 143.39 Units) respectively. Conclusion: Bacillus thuringiensis aizawai HD 865 strain is considered a low - salt tolerant strain, while the alkaline protease extracted from it is heat - resistant.