Production, purification, characterization and determination of industrial application potentials of protease from thermophilic bacteria

Objective: In this PhD thesis, it is aimed to purify and characterize the protease enzyme obtained from thermophilic bacteria and to determine the industrial application areas by increasing the production of this enzyme with recombinant DNA technology. Material and Methods: The highest protease producing isolate was determined by quantitative and qualitative protease activity determination methods among 201 thermophilic bacteria. The protease production of the selected isolate was optimized by the experimental design and strain identification was performed by the 16s rRNA method. The enzyme was purified using ammonium sulfate precipitation, hydrophobic interaction chromatography and ionic interaction chromatography. After determining the type of enzyme by chemical characterization, it was cloned into E. coli BL21(DE3) cells by chemical transformation method using the pET-30a(+) vector. Industrial application potentials of the obtained recombinant enzyme were investigated. Results: Geobacillus thermoleovorans species was identified among thermophilic bacteria and the protease enzyme it secreted was produced recombinantly in E. coli BL21(DE3) cells with a 470-fold increase. It was determined that wild and recombinant enzymes work optimally at pH 8,0 and 70 °C. Collagenase, keratinase, meat hydrolysis and destaining activities of the recombinant enzyme were determined. Conclusion: It was concluded that the protease enzyme obtained from thermophilic bacteria can be produced in high quantities by recombinant DNA technology and can be used in industrial processes with high temperatures due to in the presence of Ca+2