Molecular identification of protease producer ORSK-4 strain and determination of optimum enzyme production conditions

Abstract

A microorganism resistant to high temperatures and producing alkaline proteases was isolated from soil samples from a protein-rich region (Kırıkkale/Yahşihan). This isolate, with high protein production, was identified as ORSK-4 by determining its morphological and biochemical properties using the 16s rRNA molecular approach and the Amplified ribosomal DNA restriction analysis (ARDRA) technique employed in strain differentiation. The optimum enzyme production conditions of the strain ORSK-4 were found to be the enzyme media, 3 days of incubation, 27.0 °C, and pH 7.0. Different components were utilized to determine the effect of changing the medium content on enzyme activity. Under the optimal production conditions determined in this way, the enzyme activity of ORSK-4 was found to be higher than that of some ATCC reference Bacillus species. To purify the extracellular protease of ORSK-4, precipitation with ammonium sulfate (30% and 80%), dialysis, and DEAE ion exchange chromatography were performed. SDS-PAGE analysis determined the molecular weight of the purified enzyme as approximately 30 kDa. Although the enzyme showed activity at various pH ranges, it showed its maximum activity when increased up to pH 9.0. In conclusion, the stability of the obtained alkaline protease enzyme under different conditions shows that it can be used in industrial and environmental applications.