Determination of extracellular peptidase producing ability of rope-forming strains of Bacillus, partial optimization, purification and characterization of the peptidase

In this study, the aim was to evaluate the peptidase producing capacity of Bacillus species and whether they are suitable for industrial use. For this purpose, partial optimization, purification and characterization of the peptidases produced was performed. In the first part of the study 39 Bacillus species isolated from ropy bread were investigated by toxin kits for the production of hemolytic (Hbl) and non-hemolytic (Nhe) enterotoxins. Then, 14 of the strains which were found not to produce either of these toxins were analysed for their extracellular peptidase production capacity at 30 °C, 37 °C, 50 °C and 55 °C, by measuring the peptidase activity. Among these 14 strains, K1 and K10 which has the highest peptidase activity in unit/ml and N8 which has the highest activity in unit/ml/OD were selected for use in the production of peptidase. Peptidase activity of Bacillus subtilis PY22, B. subtilis RSK 244 and B. subtilis RSK 246 were also determined for comparison purposes. In the second part of the study, the optimum enzyme production media composition and conditions were determined for the strains K1, K10 and N8 selected by using one factor at a time method approach. For this purpose, carbon source, nitrogen source, carbon/nitrogen ratio, agitation rate and inoculum culture media which ensure the best peptidase activity were determined by considering the cost-effectiveness. The best carbon and nitrogen source was determined as glucose and nitrogen, respectively. Furthermore, better enzyme activity was obtained with a carbon/nitrogen ratio of 1:5, agitation rate of 250 rpm and use of the same media for preparing inoculum culture as for enzyme production. The third part of the study involved the optimization of peptidase production through response surface methodology using the K1 strain, which yielded the best peptidase activity in the previous part. Temperature, initial pH of media and inoculation rate were used as the factors for this process. The optimum temperature, initial pH of media and inoculation rate in terms of peptidase production were found as 33.4 °C, 6.62 and 2.3% respectively. Peptidase activity of 49.17 unit/ml was measured and specific activity of 504.77 units/mg was calculated for the crude enzyme under these conditions. In the last part of the study crude enzyme solution (peptidases) produced under the optimum conditions mentioned above were purified by affinity chromatography and characterized in terms of some of their properties. Peptidases could be partially purified with an efficiency of 25% and a purification coefficient of 1.53. It was determined that the optimum pH of partially purified peptidase solution was 7.5 and the peptidases retained approximately 90% of its initial activity between the pH range of 7.0-8.5 after an incubation at 37 °C for 2 hours. It was found that the optimum temperature for the partially purified peptidase was 60 °C, however, the enzyme mixture did retain its activity for a longer period of time at 50 °C. The inactivation of the partially purified enzyme in the presence of O-FEN and EDTA (1-4 mM) showed that the peptidases produced were metallopeptidase. Furthermore, it was determined with the use of SDS-PAGE and zymography analysis that the approximate molecular weight of the partially purified enzyme was 36 kDA. Five mM of K+1 and 5 mM of Mn+2 ions increased the enzyme activity by 4 and 6% respectively, on the other hand, Hg+2 and Fe+3 ions inactivated the enzyme. It was determined that the partially purified enzyme was inactivated in the presence of SDS (%0.1-1.0 w/v), however the enzyme retained most of its activity upon incubation at 37 °C for 30 minutes in the presence of %0.1-1.0 (v/v) Triton X-100, Tween 20, Tween 80 and %1-20 (v/v) xylene, ethanol, acetone and acetonitrile.