Method for purifying enzymes from clostridium histolyticum using multi-stage chromatography
The invention relates to a method for purifying at least one enzyme obtained in an excess fermentation of Clostridium histolyticum. It is provided for that the enzymes of the excess fermentation are separated by a multistage chromatography method by exclusively using chromatography materials on styrene/divinyl-benzene base and/or on base of in particular ceramic hydroxylapatite.
1. 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.
2. A method according to claim 1 , wherein the method exclusively comprises separations which are based on electrostatic interactions and/or on ionic bonds and/or ion complex bonds.
3. A method according to claim 1 , wherein at least one of the elutions is a stepwise elution.
4. A method according to claim 1 , wherein the method comprises a first chromatographic stage on sintered hydroxylapatite material, a second chromatographic stage on an anion-exchanger material on sytrene/divinylbenzene base and eventually a third chromatographic stage on a cation-exchanger material on styrene/divinylbenzene base.
5. A method according to claim 1 , wherein the elution in step a) is carried out at least 300 cm/h.
6. A method according to claim 1 , wherein the elution in step b) is carried out at least 1000 cm/h.
7. A method according to claim 1 , wherein the method does not comprise any protein precipation steps.
8. A method according to claim 1 , wherein pressure stable chromatography materials are used.
9. A method according to claim 1 , wherein the chromatography is carried out as column chromatography.
10. A method according to claim 1 , wherein type II collagenase with a specific activity of at least 13000 U/g is obtained.
11. A method according to claim 1 , wherein type I collagenase with a specific activity of at least 3,000 U/g is obtained.
12. A method according to claim 1 , wherein clostripain with a specific activity of at least 200 U/mg is obtained.
13. A method according to claim 1 , wherein caseinase with a specific activity of at least 1,200 U/mg is obtained.
14. A method according to claim 1 , wherein type II collagenase and/or type I collagenase and/or clostripain are obtained with a purity of at least 70%.
15. A method according to claim 3 , wherein all chromatography stages are carried out as stepwise elutions.
16. A method according to claim 10 , wherein type II collagenase with a specific activity of at least 18000 U/mg is obtained.
17. A method according to claim 11 , wherein type I collagenase with a specific activity of at least 5,000 U/g is obtained.
18. A method according to claim 12 , wherein clostripain with a specific activity of at least 300 U/mg is obtained.
19. A method according to claim 13 , wherein caseinase with a specific activity of at least 1500 U/mg is obtained.
20. A method according to claim 14 , wherein type II collagenase and/or type I collagenase and/or clostripain are obtained with a purity of at least 80%.