Purifikacija i svojstva kolagenolitičke proteaze proizvedene s pomoću Bacillus cereus MBL13

A novel collagenase-producing bacterium has been isolated and identified as Bacillus cereus MBL13. From the culture supernatant of B. cereus MBL13 grown on bone collagen as the sole carbon and nitrogen source, an extracellular protease with novel property of hydrolyzing waste animal bones was purified. The molecular mass of the purified collagenolytic protease was estimated to be (38.0±1.5) kDa. As determined by amino acid analysis, it had high contents of asparagine, lysine and serine. The optimum temperature and pH for the collagenase activity were 40 °C and pH=8.0, respectively. The results of the effects of some metal ions, inhibitors and protein substrates suggested that the purified collagenolytic protease is a member of the metalloproteases. Type I collagen (the typical collagen in animal bone) was used as the substrate for determination of Michaelis-Menten kinetics. The obtained Km value was (1.31±0.05) g/L and the corresponding vmax value was (12.54±2.5) μmol/min. The study assumes that the collagenolytic protease purified from B. cereus MBL13 strain could be applied in the hydrolysis of waste animal bones.Izolirana je nova bakterija koja proizvodi kolagenazu, identificirana kao Bacillus cereus MBL13. Iz supernatanta kulture B. cereus MBL13, uzgojene na koštanom kolagenu kao izvoru ugljika i dušika, izdvojena je ekstracelularna proteaza sa sposobnošću razgradnje životinjskih kostiju. Procijenjena je molekularna masa pročišćene kolagenolitičke proteaze od (38,0±1,5) kDa. Analiziran je aminokiselinski sastav i utvrđeno da enzim sadržava veliku količinu asparagina, lizina i serina. Optimalna temperatura za aktivnost kolagenaze bila je 40 °C, a optimalna pH-vrijednost 8,0. Prema utjecaju iona metala, inhibitora i proteinskih supstrata na aktivnost enzima zaključeno je da enzim pripada u grupu metaloproteaza. Kolagen tipa I (tipični sastojak životinjskih kostiju) upotrijebljen je kao supstrat pri određivanju parametara Michaelis-Mentenove kinetike. Km vrijednost bila je (1,31±0,05) g/L, a vmax (12,54±2,5) μmol/min. Zaključeno je da se kolagenolitička proteaza izdvojena iz B. cereus MBL13 može upotrijebiti za razgradnju životinjskih kostiju