Fifty three local bacterial isolates of a thermophilic Bacillus spp. Were obtained from 93 soil samples taken from different locations over Basrah governorate (Iraq). All these isolates were subjected to identification by studying their morphological and microscopical and biochemical characteistics. Results showed that 6 of these isolates belong to Bacillus stearothermophilus. The local six B. stearothermophilus were screened according to their ability of protease production. Results showed that all these isolates were protease producers. Isolate B. stearothermophilus B17 was the most efficient in production when the specific activity of protease in its crude filtrate was 36.4U/mg protein. Enhancement of protease production in the locally isolated B17 was achieved by two methods: First. Cloning of protease gene in a cloning vector, and the recombinant molecules were used to transform suitable host. This was achieved first by amplification of protease gene from B17 using polymerase chain reaction (PCR) technique, then the amplified fragments coding for protease were cloned into pJET blunt cloning vector, and the recombinant molecule was used to transform Escherichia coli DH5α deficient in protease production (Pr–). Results showed that many transformants of E. coli were able to make a halo of hydrolysis around each clone on skim milk agar medium after incubation at 37°C for 24hours. Specific activity in the crude filtrate of the most efficient clone (symboled DH5α5) in protease production was 42 U/mg protein. In another attempt for cloning protease gene from B. stearothermophilus, genomic DNA of B17 was isolated, partially digested with Sau3A, and ligated to BamHΙ digest of pUC18. The recombinant molecules were used to transform E. coli DH5α (Pr–), then from about 2000 ampicillin resist transformants, 6 clones (0.3%) were able to form a halo of hydrolysis on skim milk agar plates due to their ability in protease production. The specific activity of protease in crude . filtrate of the most efficient clone (symboled DH5αP7) was 39.5 U/mg protein Second. Subjection to random mutagenesis using physical and chemical mutagens. This was achieved by subjection of the wild type B17 to physical mutagen using UV ray under different doses of irradiation (1, 2, 3, 4, 5 and 6 Jole/m2). Results showed that 6 mutants out of 45 (13.3 %) were able to make a halo of hydrolysis on skim milk agar plates after incubation at 55°C for 24 hours. These mutants were screened for protease production, and it was found that all mutants were protease producers; the specific activity of protease in crude filtrate of the most efficient over producer mutant (B17U1) was 80.08 U/mg protein in comparison with 36.4 U/mg protein for the wild type B17. Chemical mutagenesis was also used to enhance the ability of B17 in protease production by incubation with 1-methyl-3-nitro-1-nitroso-guanidine (MNNG) in a concentration of 200 μg/ml for different periods of time (20, 40, 60, 80, and 100 minutes). Results showed that 3 mutants out of 32 (9.3%) were able to make a halo of hydrolysis on skim milk agar plates after incubation at 55°C for 24h. After screening the mutants for protease production, all of these mutants were protease producers; the specific activity of protease in culture filtrate of the most efficient over producer mutant (B17M1) was 99 U/mg protein in comparison with 36.4 U/mg protein for the wild type B17. Optimum conditions for protease production by the most efficient over producer mutant B17M1 was studied, and it was found that cultivation of this mutant in the production medium containing 1% of each of starch and yeast act, pH 7, for 18h. at 55°C gave higher productivity of protease when the specific activity of enzyme in crude filtrate that reached 99 U/mg protein. Protease produced by the most effecient mutant under the optimum conditions was purified by several steps including precipitation by ammonium sulfate (80% saturation), dialysis, affinity hromatography using Bacitracin-silica column, and finally gel filtration using Superdex-75 column with 28.3 fold of purification and % 42.5 yield. Result of characterization of the purified enzyme showed that the molecular weight of protease was 31,000 dalton; the enzyme was active at pH 7, and stable within a pH range 6-7, active at 60°C, stable till 70°C for 30 min. Results also showed that protease produced by B. stearothermophilus B17M1 was a metalloenzyme because its enzyme activity was completely inhibited after incubation with 2mM EDTA.
