Purification And Characterization Of A Collagenase From Marine Microorganisms

Collagen mainly exists in animal bones and skins as one of the important proteins in organisms, which is an important structural protein in connective tissue and plays the role as a material basis. It can support and protect the organs, which is the life scaffold for human body and other animals. Collagen was absorbed by human body when it is degraded into small molecular peptides and amino acids. But its unique triple helical structure is difficult to be destroyed, therefore most edible collagen are difficult to be directly and effectively used by human body. After the collagen is degraded by enzyme into peptides, it will be easy to be absorbed by human degraded. It also have many significant functions of protection of gastric mucosa, anti-ulcer effect, protection of high blood pressure and promoting bone formation as well as the improvement of skin. At the same time, it will have good prevention and treatment effect on collagen diseases such as arthritis etc.The most effective method to hydrolyze collagen is to use collagen enzyme. Collagenase is defined as the enzyme that only cut the helical regions of activated collagen or other protein gelatin under the appropriate pH and temperature, but does not have any effect on other protein substrates. At present, collgaenolytci protease has been obtained from many animal tissues and microorganisms.This study optimized the composition of culture medium and fermentation conditions for producing strains provided by our laboratory to produce collagenase. The gelatin was made as the inducer of the producing strain. And the producing strain proliferated largely after the liquid fermentation. The folin-phenol method was used to determinate the enzyme activity. The results shown that the enzyme activity in the fermentation supernatant reached up to50.68U/mL. The collagenase was separated and purified by the steps of centrifugation, ammonium sulfate precipitation, dialysis and sephadex G-75gel chromatography. The enzyme activity could reach up to19.87U/mg, the purification multiple was8.8and the yield was0.38%. The molecular weight of the collagenase was54kDa detected by SDS-PAGE electrophoresis. And the enzymology properties of the collagenase produced by the bacterial strain were studied. The results shown that the optimal reaction temperature for collagenase was40℃, the optimal pH was8and Mn2+could promote bacteria growth as well as Cu2+. The low concentration of Fe2+could also promote bacteria growth, but high concentration of Fe2+inhibit the bacteria growth. Mn2+and Fe+had inhibited the enzyme activity. The relative enzyme activity was5.2%when the concentration of Fe2+was5mmol/L. Low concentration of Cu2+could promote enzyme activity. When the concentration of Cu2+reached to2mmol/L, the relative enzyme activity reached149.01%.But it changed to a inhibitor of the enzyme under high concentration.