Abstract: The identification of the most sensitive sites of Clostridium histolyticum collagenase Class 1 to proteolysis by proteases present during the fermentation and purification of the enzyme is described. Culture supernatant obtained after fermentation of C. histolyticum is used as the starting material for further purification of the enzyme. Native collagenase Class 1 and its proteolytic fragments are partially purified by a combination of hydrophobic interaction and strong anion exchange chromatographies. The pools containing enriched levels of the proteolytic fragments are further purified by high performance anion exchange chromatography. These polypeptides are then characterized by Q-TOF mass spectroscopy. A total of three sensitive bonds are identified along with substitution and deletion strategies that will result in resistance of the enzyme to proteolytic degradation.

Abstract: A purified enzyme mixture useful for isolating cells or cell clusters from tissue is disclosed. The mixture includes at least two collagenase enzymes, at least two other proteases, and additional non-protease components. The mixture is purified by removing at least some of the non-protease components. The purified mixture may then be used to isolate cells or cell clusters from tissue. Also disclosed are the essential components of the purified enzyme mixture as well as preferred ranges and ratios of these essential components for isolating cells or cell clusters from tissue. Finally, a dissociation system is disclosed that can be used with the purified enzyme mixture to dissociate tissue and recover cells or cell clusters.

Abstract: An enzyme composition and its use for isolating cells or cell clusters from tissue is disclosed. The composition includes two purified collagenase enzymes and an endoprotease. The ratio of the total FITC casein activity of the enzyme composition to the Wunsch units of activity of the masses of collagenase I and collagenase II is about 85:1 to about 3,900:1.

Abstract: An enzyme composition for dissociating tissues is disclosed. The composition comprises purified collagenase I and collagenase II from Clostridium histolyticum in a mass ratio of about 0.3 to 0.6 and a neutral protease such that the ratio of total FITC casein units of activity to Wunsch units of activity in the composition is about 250:1 to about 600:1. The preferred neutral protease is thermolysin.

Abstract: A purified enzyme mixture useful for isolating cells or cell clusters from tissue is disclosed. The mixture includes at least two collagenase enzymes, at least two other proteases, and additional non-protease components. The mixture is purified by removing at least some of the non-protease components. The purified mixture may then be used to isolate cells or cell clusters from tissue. Also disclosed are the essential components of the purified enzyme mixture as well as preferred ranges and ratios of these essential components for isolating cells or cell clusters from tissue. Finally, a dissociation system is disclosed that can be used with the purified enzyme mixture to dissociate tissue and recover cells or cell clusters.