Method for purifying enzymes from clostridium histolyticum using multi-stage chromatography

A method of purifying caseinase, type I collagenase, type II collagenase and clostripain produced in a medium by the fermentation of Clostridium histolyticum, comprising:

• a) substantially separating caseinase from the other three enzymes by exposing the medium to a sintered hydroxyapatite chromatographic material followed by eluting the enzymes at flow rates of at least 200 cm/h such that fractions are eluted comprising caseinase substantially separated from the other three enzymes and fractions are eluted which are substantially free of caseinase but contain the type I collagenase, type II collagenase and clostripain;
• b) substantially separating the type I and type II collagenases by exposing the fractions which are substantially free of caseinase but contain the type I collagenase, type II collagenase and clostripain to an anion-exchange chromatographic material with a styrene/divinylbenzene base and eluting the enzymes, at a flow rate of at least 500 cm/h, such that a fraction is eluted comprising type I collagenase and clostripain but substantially free of type II collagenase and a fraction is eluted comprising type II collagenase and clostripain but substantially free of type I collagenase;
• and
• c) separately exposing the type I collagenase/clostripain fraction and the type II collagenase/clostripain fraction to cation exchange chromatography and eluting the enzymes such that separate fractions are eluted comprising type I collagenase substantially free of clostripain, type II collagenase substantially free of clostripain and clostripain substantially free of either collagenase,
• wherein steps a), b) and c) are all carried out at between 4 and 25° C.