Production, Purification, and Some Properties of Clostridium histolyticum Collagenase.∗

Jennison(1) reported that Clostridium histolyticum produced a proteinase that was able to digest “native” collagen. In a previous communication from this laboratory(2) the activity of Cl. histolyticum filtrates in the degradation of collagen was confirmed, as well as the lack of activity of other proteinases on unmodified collagen. In this communication a reproducible procedure will be described for the isolation and partial purification of a proteinase(s) from Cl. histolyticum which is very active in the solubilization of native collagen. A method will be described wherein filtrates with little or no lethal toxin but adequate “collagenase” activity may be produced in large batches. The final product purified by the method to be described is 400 times as active per unit nitrogen as the original filtrate. The separation of a proteolytic enzyme which has no collagenase activity and which is activated by Fe and cysteine (gelatin substrate) is described. This enzyme has been described previously (3,4). Experimental. Stock and seed cultures. The strain of Cl. histolyticum used in these studies was CHT from the stock collection in this laboratory. The stock medium was prepared as previously described for Cl. perfringens(5). In carrying this culture on stock medium, no glucose was used. Just before transfer, 1 ml of a sterile sodium thioglycolate solution (4 mg) was added to each tube. The stock cultures were incubated at 37°C for 16–18 hours. Seed cultures were incubated for 8 hours. Medium for collagenase production. Casein digest and medium were prepared as previously described(5). The bottles containing 3 liters of medium were steam sterilized at 15 lb. for 1 hour and cooled to approximately 38–40°C in running tap water. A sterile solution (75 ml) containing 3 g of sodium thioglycolate was added to each bottle immediately prior to incubation with 40 ml of seed culture.