Patent ID: 7083964

Claim:
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.