Collagenase

Collagenase can specifically hydrolyze the three-dimensional helical structure of native collagen under physiological pH and temperature conditions without damaging other proteins and tissues. The chemical nature of collagenase is a protein, and therefore, it is very sensitive to temperature, pH, and various factors that cause protein denaturation, and is easily influenced by external conditions that can change its conformation and properties.

Classification

Collagenase can be divided into human endogenous collagenase and medicinal collagenase according to their different states of existence. Human endogenous collagenase refers to the collagenase that exists in the human body. There are different levels of collagenase in the epithelial tissues, joint synovium and intervertebral disc. It plays an indispensable role in the decomposition of collagen in the body. The medicinal collagenase refers to a white or white-like aseptic freeze-dried powder obtained by extracting, purifying, and purifying from the fermentation broth of Clostridium histolyticum using high-tech means of biopharmaceuticals.

Collagenase can also be divided into class I, II, III, IV, V and special collagenase for liver cells. The type of collagenase should be selected according to the type of tissue to be separated and digested. Collagenase I is used for the separation of epithelial, lung, fat and adrenal tissue cells. Collagenase II is suitable for liver, bone, thyroid, heart and salivary gland tissue. Collagenase IV contains at least 7 protease components with molecular weights ranging from 68 to 130KD. It can digest a variety of tissues. Collagenase V can be used for the isolation of pancreatic islet tissue, which separates connective tissue into single cells.

Structure

Collagenase is produced by two separate and distinct genes in Clostridium histolyticum. Both genes have been cloned and sequenced. The colG gene codes for type I collagenase, a 936 amino acid peptide. The colH gene codes for type II collagenase, a 1021 amino acid peptide. These genes share 72% identity, and the proteins share 43% identity. Both gene products can be present as two or more isoforms differing in molecular weight. Crude collagenase mixtures can therefore contain six to eight different molecular weight species ranging from 68 to 130 kDa. Substrate specificity studies have demonstrated that the colG gene prefers natural substrates such as intact collagen, compared to the colH gene product. Conversely, the colH gene product preferentially acts on short synthetic substrates (FALGPA) relative to the colG gene product.

Properties

The optimum pH of collagenase is 6 to 7, the optimum temperature is 35 to 45°C. The enzyme activity is stable below 45°C, and the activity is irreversibly lost above 80°C for 10 minutes. Metal ions, such as Ca²⁺, Mg²⁺, Ba²⁺, Mn²⁺, can promote enzyme activity, while Co²⁺ and Hg²⁺ can inhibit enzyme activity. After complete dialysis of Ca²⁺, the activity of collagenase is not lost. After 20 mmol/L of EDTA is added, and the enzyme activity almost disappears. An additional excess of calcium chloride can only restore 85% of the activity. Collagenase activity is inhibited by the metal ion chelator EDTA, the mercapto compounds (mercaptoethanol, reduced glutathione) and normal human plasma.

Catalytic Mechanism

The hyper-reactive sites where the Class I and Class II enzymes initially attack all three collagen types were identified by scientists in 1992. The cleavage sites are all at Yaa-Gly bonds in the repeating Gly-X-Y collagen sequence. Clostridial collagenases’ ability to digest native, triple-helical types I, II, and III collagens into a mixture of small peptides is its primary distinguishing factor. This is accomplished by making multiple scissions in the triple helix. Digestion is completed by hydrolyzing those fragments into a mixture of small peptides. Conversely, vertebrate collagenases initiate collagenolysis by making a single scission across all three alpha chains after which attack on those alpha chains is very limited. Gelatinases and other proteases then carry out collagenolysis only after denaturing of the triple helix.

Applications

Collagenase extracted from bacteria is mainly used in clinical and laboratory applications. Clinical applications include trauma therapy, treatment of lumbar disc and sciatic nerve, etc. In the laboratory, collagenase is mainly used for the isolation of liver cells in mice and the degradation of collagen. In addition, collagenase can also tenderize meat by degrading collagen, which is of great significance in the food industry.