Collagenase from Clostridium histolyticum

Collagenase from Clostridium histolyticum has been used in determining the degradation rate of collagen biomaterials. It may also be used to determine the enzymatic degradation and enzymatic resistance by the liberated L-leucine measurements.

Collagenase from Clostridium histolyticum, or Clostridiopeptidase A, has been used in a study to assess contact dermatitis with clostridiopeptidase A contained in Noruxol ointment. Clostridiopeptidase A has also been used in a study to investigate the early and successful enzymatıc debridement via collagenase application to pinna in a preterm neonate.

The enzyme has been used in the isolation of mast cells from adult mice. This study generated a large number of connective tissue-type mast cells by the culture of murine fetal skin cells. The product has also been used in the in vitro biodegradation study of Bombyx mori silk fibroin fibers and films, by exposing them to the enzyme for varied time periods.

Effective release of cells from tissue requires the action of collagenase enzymes and the neutral protease. Collagenase is activated by four gram atom calcium (Ca²⁺) per mole enzyme. The culture filtrate is thought to contain at least 7 different proteases ranging in molecular weight from 68-130 kDa. The pH optimum is 6.3-8.8. The enzyme is typically used to digest the connective components in tissue samples to liberate individual cells. Ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA)4; β-mercaptoethanol; glutathione, reduced; thioglycolic acid, sodium; and 2,2′-dipyridyl; 8-hydroxyquinoline are known to inhibit the enzyme activity.

Collagenase is activated by four gram atom calcium per mole enzyme. It is inhibited by ethylene glycol-bis(beta-aminoethyl ether) - N, N, N′,N′-tetraacetic acid, beta-mercaptoethanol, glutathione, thioglycolic acid and 8-hydroxyquinoline.

One collagen digestion unit (CDU) liberates peptides from collagen from bovine achilles tendon equivalent in ninhydrin color to 1.0 μmole of leucine in 5 hours at pH 7.4 at 37 °C in the presence of calcium ions. One FALGPA hydrolysis unit hydrolyzes 1.0 μmole of furylacryloyl-Leu-Gly-Pro-Ala per min at 25°C. One Neutral Protease unit hydrolyzes casein to produce color equivalent to 1.0 μmole of tyrosine per 5 hr at pH 7.5 at 37°C. One Clostripain Unit hydrolyzes 1.0 μmole of BAEE per min at pH 7.6 at 25°C in the presence of DTT.