Isolation of keratinolytic Bacillus sp. strains, keratinase production and characterization

In this study, twentyone proteolytic (on skim-milk agar) Bacillus strains were isolated from poultry soil. Eight strains of these bacteria were showed keratinolytic activity when keratine azure used as substrat.HSK-21 strain was chosen the best keratinase producer and identified as Bacillus sp. via VITEK-2 (bioMerieux) bacteria identification system.The basal feather medium was used for keratinase production and maximum enzyme activity measured at pH=8.0 and 30ºC.The enzyme was active at pH 5.0-13.0 and at 20-60ºC, though optimum activity was obtained at pH=12.0 and 40ºC.When the enzyme tested at different buffers (pH 7.0-13.0) for 30 sec., 50-100% activity remained and the activity (100%) was stable at pH=11.0 and pH=12.0. When the enzyme incubated at 20-100ºC for 30 sec., 34-86% activity retained at 20-60ºC, while 100% activity was seen at 30ºC.Three different (46,8 kDa, 26 kDa and 21.9 kDa) proteolytic activity protein bands were revealed with SDS-PAGE (with gelatin).After incubation for 30 sec. with enzyme inhibitors and metal ions (1-5 mM), Na2SO3 (%42), CaCl2 (%39), PMSF (%34), Phenyl Glyoxal (%23), urea (%15) induced enzyme activity and N-Bromo süksinimide and ZnCl2 inhibited completely. While HgCl2 (%66), SDS (%56), EDTA (%46), Ethyl acetimidate (%32), MgCl2 (%15) decrased activity, KCl ve NiCl2 didn?t cause any effect. The wool were succesfully dehaired from sheep leather pieces at pH=12.0 and 40ºC.The results showed that, the keratinase from Bacillus sp. HSK-21 is useful for mesophilic and highly alkaline conditions in biotechnological applications requiring in keratinolisis and the enzyme was the only bacterial keratinases working high pH conditions (optimum pH=12.0) documented in the literatures so far.Key Words: Bacillus sp. HSK-21, alkaline keratinase, dehairing