title: Purification and characterization of a novel collagenase from Bacillus pumilus Col-J.
abstract: The collagenase, produced extracellular by Bacillus pumilus Col-J, was purified by ammonium sulfate precipitation followed by two gel filtrations, involving Sephadex G-100 column and Sepharose Fast Flow column. Purified collagenase has a 31.53-fold increase in specific activity of 87.33 U/mg and 7.00% recovery. The collagenase has a relative molecular weight of 58.64 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimal temperature for the enzyme reaction was 45 degrees C. More than 50% of the original activity still remained after 5 min of incubation at 70 degrees C or 10 min at 60 degrees C. The maximal enzyme activity of collagenase was obtained at pH 7.5, and it was stable over a pH range of 6.5-8.0. The collagenase activity was strongly inhibited by Mn(2+), Pb(2+), ethylenediamine tetraacetic acid, ethylene glycol tetraacetic acid, and beta-mercaptoethanol. However, Ca(2+) and Mg(2+) greatly increased its activity. The collagenase from B. pumilus Col-J showed highly specific activity towards the native collagen from calf skin. The K(m) and V(max) of the enzyme for collagen were 0.79 mg/mL and 129.5 U, respectively.
authors.affiliation: College of Life Science, Sichuan Agricultural University, Ya'an, China. wuqiwq@yahoo.cn
authors.forename: Qi
authors.initials: Q
authors.lastname: Wu
authors.forename: Chen
authors.initials: C
authors.lastname: Li
authors.forename: Chenglei
authors.initials: C
authors.lastname: Li
authors.forename: Hui
authors.initials: H
authors.lastname: Chen
authors.forename: Liu
authors.initials: L
authors.lastname: Shuliang
mesh_terms: D000818:Animals; D001407:Bacillus / Q000201:enzymology* / Q000302:isolation & purification; D002417:Cattle; D017364:Collagenases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D017077:Culture Media, Conditioned / Q000378:metabolism; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents / Q000494:pharmacology; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D017077:Culture Media, Conditioned; D007202:Indicators and Reagents; D008670:Metals; D017364:Collagenases