The Isolation of Collagenase and Its Enzymological and Physico-chemical Properties

A collagenolytic enzyme specific for native collagen and gelatin was isolated from Pseudomonas marinoglutinosa by DEAE-cellulose column chromatography, Sephadex G–150 gel filtration and by disc electrophoresis on polyacrylamide gel.

The molecular weight of the enzyme was approximately 74,000 and its isoelectric point was found to be around 4.5. The optimum pH and temperature for Z–GPLGP hydrolysis were around 7.6 and 38°C, respectively. The enzyme was rather stable up to 50°C and in the range between pH 5.0 and 10.0, and was stabilized by Ca²⁺ to some extent. Some chelating agents and metal ions such as Hg²⁺, Pb²⁺, Zn²⁺, Ni²⁺ and Fe²⁺ inactivated the enzyme, but diisopropyl phosphofluoridate, sulfhydryl agents and some trypsin inhibitors did not affect the activity.

The EDTA-inactivated enzyme was restored its activity by added Ca-salt to almost completely and very slightly by Co-, Mn- and Sr-salt.

Metal analysis showed the enzyme contained 1 g atom of zinc and 4 g atoms of calcium per mole.