Process for purifying collagenase

Details Process for purifying collagenase Process for purifying collagenase

1993-04-16

1994-07-26

Therkorn, Ernest G.

Liquid purification or separation

Processes

Liquid/liquid solvent or colloidal extraction or diffusing...

210656, 435 41, 435 703, 435219, 435220, 435226, 4352527, 435815, 435842, B01D 1508

Patent

active

053325036

ABSTRACT:
A process for purifying crude collagenase is disclosed. The collagenase purification process includes providing a stabilized crude collagenase solution containing collagenase, pigment, toxins, bacterial materials, and proteolytic enzyme impurities including clostripain, trypsin, and caseinase. The stabilized collagenase solution is applied to hydroxylapatite packing. pigment and caseinase are eluted with a first solution comprising about 0.05 M to about 0.3 M phosphate buffer, and then collagenase, trypsin, and clostripain are eluted with a second solution comprising about 0.35 M to about 0.5 M phosphate buffer to provide a first collected solution. The first collected solution is then applied to gel filtration packing and collagenase and clostripain are eluted with a neutral pH buffer solution, to provide a second collected solution. The second collected solution is then applied to Reactive Red 120-Agarose packing and collagenase is eluted with a neutral pH buffer solution to provide purified collagenase. The process provides extremely pure collagenase in high yield with reduced consumption of eluting solutions and avoids unpredictable gradient eluting techniques.

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Bond and Van Wart, "Purification and separation of individual collagenases of clostridium histolyticum using red dye ligand chromatography", Biochemistry 1984, 23, 3077-3085.
Abstract of France Patent No. 2366306 from Food, Disinfectants, Detergents, Week A26, p. 6, 1978.

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