Studies on the protease enzyme in Bacillus cereus KG5

In this survey it is aimed to study the extracellular protease enzyme which has a wide application in the industry of Bacillus cereus KG5 which has been isolated in Kös (Bingöl) hot spring.B.cereus KG5 BM has taken under culturization at thee feed-lot and protease activity is determined at various incubation times and the maximum enzyme production is determined at the 24th hour.The effect of pH and temperature over the protease enzyme is studied under pH 6-11 and t between 20°C and 70ºC temperatures. It was determined that the optimum value of pH and temperature for the enzyme is respectively 7.0 and 40-45ºC. The effects of different feed-lots and %1.2 percent different nitrogen and %2 percent carbon sources has been studied. The maximum enzyme production is acquired at BM feed-lot. It is determined that the best nitrogen source is yeast ekstrakt and urea, the best carbon source is lactoz and galaktoz meanwhile glukoz as a source of carbon inhibated the production of the enzyme. Different consantrations of yeast effect over enzyme production is studied. The maximum enzyme production is acquired at %0.5 yeast extract. It is observed that the enzyme production is degraded by yeast extract increase. Different metal ions effect at the ratio of %0.5 percent over enzyme production is studied. The CaCl2 approximately doubled the enzyme production, NaCl ve MgCl2 dramatically reduced the enzyme production. CaCl2?s effect over enzyme production is studied. The maximum enzyme production is acquired at %0.5, and the lowest enzyme production is acquired at %0 CaCl2 consantrations.Some metals, chemicals, metal chelat agents and detergents effects over protease enzyme activity that is belonging to partially purified B.cereus KG5 is studied. CaCl2 (%142 at 2 mM ) , MgCl2 (%89 at 5 mM and 10 mM) and MnCl2 (%29 at 2 mM) increased the protease activity at a certain extent, CuCl2, HgCl2 and ZnCl2 (at 10 mM) inhibated the enzyme activity respectively at %100, over %100 and %96, EDTA and 1-10 phenantroline which are metal chelat agents (respectively %96 and %95 at 10 mM) heavly inhibated the protease enzyme. At the effect of PMSF, an inhibition due to etanolet has been determined. It is determined that the enzyme activity is inhibated at % 1 SDS fully, %1 Alo at %84 percent, % 0.5 Triton X-100 at % 5 percent and 0.1 Tween-80 at % 2 percent . As a result of studying the thermal stability of the enzyme, it is determined that it is stil stable at 40ºC temperature at the end of 120 minutes. İt is determined that the thermal stability of the enzyme is increased by CaCl2 . It is determined that 2 mM CaCl2 enzyme at 50ºC temprerature at the end of the 120 minutes still preserved the original stability at % 102 percent.In this study, the protease of B.cereus KG5 was purified by amonium sulfate precipitation&dialysis and Sephadex G-75 gel permeability chromatography with 13 fold and %23 recovery.The enzyme purified by the Sephadex G-75 gel permeability chromatography existence is determined by nondenaturing polyacrilamide gel electrophoresis meanwhile the molecule weight is determined approximately 48 kDa by SDS-PAGE.Key words: Bacillus cereus KG5, Biotechnology, protease enzyme production and characterization.