Isolation and identification of Thermophilic bacteria isolated from hot water samples collected in Van province, Turkey and purification-characterization of protease enzyme from Bacillus licheniformis EA10 isolate with three phase partitioning system

In this thesis work, a total of 10 thermophilic bacteria were isolated from hot water samples collected from Van Çaybağı and Hasanabdal springs. 10 isolates were identified as follows using conventional and molecular analysis; EA1, EA2, EA4, EA5, EA6, EA7: Bacillus thermolactis; EA3, EA9, EA10: Bacillus licheniformis; EA8: Anoxybacillus pushchioensis. According to a pedri analysis, three strains were observed to be positive in terms of protease activity and EA10 had the highest activity. Purification of protease from B. licheniformis EA10 using TPP was carried out for the first time in this study to the best of our knowledge. For this, optimization of salt content, organic solvent and pH was done. The TPP system applied at pH 6.0 using 1.0: 0.5 (crude enzyme solution: t-butanol ratio) and 70% saturated ammonium sulphate resulted in 54.1 fold of enzyme purification with 191.6% yield and the molecular weight of the purified enzyme was estimated to be approximately 37.67 kDa by SDS-PAGE gel electrophoresis. The optimum pH and temperature were determined as 9.5 and 80 °C, respectively. It was found that the enzyme did not decrease its activity at all after 120 minutes of incubation at a temperature as high as 90 °C and that the incubation period had an activity-enhancing effect. It was also found that B. licheniformis EA10 protected its protease enzyme stability by 107% and 102% in the presence of H2O2, an oxidizing agent, at concentration ratios of 1% and 5%, respectively. The enzyme exhibiting high stability against organic solvents maintained its activity at 1 mM Mn2+ and increased its activity by 164 and 237% at 5 and 10 mM concentrations, respectively. In the presence of EDTA, enzyme stability was preserved by 104.6%. Results showed that alkaline protease of B. licheniformis EA10 had high substrate specificity for both casein and gelatin and the KM and Vmax values for casein substrate was calculated (KM; 0,376 mg/ml and Vmax; 14,085 µmol.ml-1.dk-1 ).