Isolation, characterization, and investigation of biotechnological potential of alkaline protease enzymes with different thermal stability from native and mutant Bacillus sp. strains

In this thesis study, psychrophilic, mesophilic, and thermophilic alkaline extracellular protease enzyme producer natural Bacillus sp. strains were isolated. The isolated strains were identified biochemically and molecularly. The strains were developed by random mutagenesis (physical, chemical, and combined) applications to increase enzyme efficiency. The protease production environments of the developed mutant strains were optimized and enzyme production was carried out. Protease enzymes produced from natural and mutant strains under optimum enzyme synthesis conditions were partially purified and then characterized comparatively. In characterization studies, the optimum pH and temperature values, pH and temperature stabilities, stabilities in the presence of various chemicals, and substrate specificities of the enzymes were determined. In addition, casein hydrolysis products were determined by thin-layer chromatography and HPLC analyses. After characterization, comparative applications were made using natural and mutant enzymes to determine the potential of using natural and mutant enzymes in different industrial areas. Psychrophilic, mesophilic, and thermophilic alkaline protease-producing strains were identified as Bacillus mobilis TK31, Bacillus cereus TK16, and Bacillus subtilis PTK56, respectively, by 16s rRNA analysis. The pH and temperature values at which psychrophilic, mesophilic, and thermophilic enzymes show optimum activity are 9.0 and 10ºC; 10.0 and 40ºC; 9.0 and 55ºC, respectively. As a result of random mutations, the highest yield was obtained for Bacillus mobilis TK31 and Bacillus cereus TK16 strains combined; and for Bacillus subtilis PTK56 strain, it was obtained with chemical mutation application. With the optimization of enzyme production media, the activity of the psychrophilic 18C-179 mutant enzyme (0.289 U/mL) was 3.75 times higher than the wild-type control (0.077 U/mL); the activity of the mesophile 76C-3 mutant enzyme (0.209 U/mL) was increased 1.83-fold compared to the wild-type control (0.114 U/mL) and the activity of the thermophile 56E-67 mutant enzyme (0.274 U/mL) was increased 1.34-fold compared to the wild-type control (0.201 U/mL). It has been determined that the isolated enzymes have the potential to be applied in different biotechnological fields. Keywords: Alkaline Protease, Psychrophile, Mesophile, Thermophile, Random Mutagenesis, Strain Development