Isolation And Enzyme Characteristics Of A Strain Producing Collagenase

Collagen presents in the animals’ skin, bones, cartilage, teeth, muscles, ligaments, blood vessels. It’s an important kind of structural protein which exists in the connective tissue and plays a part in supporting organs and protecting bodies. However, because the collagen has a unique triple helical structure which has a very stable character, it’s not easy to be digested and fully utilized. If the collagen’s triple helical structure could be hydrolyzed to collagen polypeptides, its efficiency would be significantly improved in many aspects such as digestion, absorption and nutrition.According to domestic and foreign studies, collagen polypeptide formed by hydrolysis of collagen possess ability of protecting the gastric mucosa, anti-ulcer, inhibiting the rise in blood pressure, promoting bone morphogenetic, accelerating skin collagen metabolism, etc. It has a huge development space in the fields of food and medicine. The most effective way to hydrolyze collagen is using collagenase. Collagenase is a kind of enzyme which works on collagen and modified gelatin rather than the other kinds of protein. Collagenase extracts from Clostridium histolyticum and Vibrio alginolyticus has been studied extensively. At present, collagenase can be acquired in many animal tissues and microbe.In this study, with gelatin as inducer, after preliminary screening, a single colony of DL-21which could produces transparent circle on nutrient gelatin panel would be isolated from ocean mixed bacterium which was provided by laboratory. After multiplied by liquid fermentation, skin hydrolysis test proved the DL-21can effectively produces extracellular collagenase. It’s a strain which has collagenase productivity. The activity of the supernatant liquid can reach up to22.46U/mL which results from Folin-phend method.We evaluated DL-21by classical methods:visual observation of colony morphology, as well as in the culture medium on the plate in the slant medium and liquid medium, the growth of state; electron microscopy of cell morphology, size, arrangement; to verify the strain of movement, special structure, Gram stain reaction; determination of strains of the ecological characteristics such as growth temperature, and oxygen; determination of strains of physiological and biochemical reactions. The results showed that the strain was Serratia entomophila. To verify the accuracy of classical methods, after16S rDNA sequencing determination and BLAST search, we find that strain DL-21has the most homology with the Serratia genus (of Serratia) in the16S rDNA sequence. The classical identification methods were verified. Construct phylogenetic tree by16S rDNA sequence from DL-21and the other eight serratia strains, we can find the result shows that the DL-21and Serratia marcescens (Serratia marcescens AU1209) and Xenorhabdus nematophila (Serratia nematodiphila DZ0503SBS1) has the closest relatives.By centrifugation, ammonium sulfate precipitation, dialysis, Sephadex G-75gel filtration chromatography, collagenase was isolated and purified. We proved the collagenase has got a fine purification after detected monochrome strip by SDS-PAGE. The relative molecular weight is57.5KDa.The preliminary study of Collagenase’s nature showed that the optimum reaction temperature is40℃and pH is8. Besides that, calcium ion, magnesium ion and copper ion could play roles in activate collagenase.