Purification And Characterization Of A Collagenase From Bacillus Pumilus

Forty-nine microbes with collagenase activity were isolated from tannery, poultry and abattoir samples in the study. The supernatant of two strains, named Col-C and Col-J, could digest the calf skin obviously and completely. Based on the polyphasic studies, including their phenotypic, physio-biochemical characteristics, 16S rDNA phylogenetic analysis, Col-C and Col-J were identified as Serratia marcescen and Bacillus pumilus, respectively.Collagenase from the supernatant of Bacillus pumilus Col-J was purified. Ammonium sulfate was added to 30% saturation and then 75% in supernatant. And the precipitate was collected by centrifugation,then the pellet was dissolved and the fraction was dialyzed. The material was then applied to gel filtration chromatography on a Sephadex G-100 column, Fraction showing collagenase activity were cellected then was put on a ion-exchange chromatography on Sepharose Fast Flow after being concentrated. Keep the active fraction Q3. SDS-PAGE showed that the molecular mass of the electrophoresis pure collagenase was about 58.64kD, The results of this experiment showed the purified-fold was 31.53, the recovery was 7.00%, respectively.The enzymatic characters studies of the purified collagenase showed its optimal temperature and pH were 45℃and 7.5, respectively. The purified collagenase showed stability between 30℃and 65℃, as well as between pH6 and pH8.5, which both can maintain over 60% of the total activity.50mmol/L Li⁺, K⁺, Zn²⁺, Mg²⁺, Ca²⁺ and Ba²⁺ were found to have some activation to collagenase, whereas, 50mmol/L Na⁺, Mn²⁺, Fe³⁺, Pb²⁺ and 0.1mmol/L EDTA, EGTA, mercaptoethanol showed some inhibition to collagenase. 50mmol/L Ca²⁺ and 0.1mmol/L EGTA showed the most obvious activation and inhibition. The relative activity were found to come to 127% and 10.1%, respectively. The purified collagenase showed specificity to collagen protein, he kinetic parameters of the purified collagena were measured at pH7.5 and 45℃using the insoluble collagen type III, K_m and V_max of the enzyume were determined to be 0.79 mg/mL and 0.1295mg/(min·mL), respectively. Comparison of properties with the other collangenases reported, the results indicated the collangenase from Bacillus pumilus Col-J is a novel one.