Comparative Study On Characteristics And Self-assembly Properties Of Collagen Extracted From Different Sources And Immobilization Of Catalase

Collagen molecules could self-assemble into fibrous structure under appropriate conditions,which could be used as a kind of biomaterials and functional materials.There were some differences in the structure and properties of collagens between various sources,which may caused the differences of self-assembly behavior and structure.Collagens were extracted from the pig skin,bovine skin and tendon,grass carp skin and scale,crucian carp skin,respectively.Their characteristics and self-assembly properties were analyzed by Ultraviolet(UV),Sodium dodecyl sulphate-polyacrylamide gel electrophoresis(SDS-PAGE),Fourier transform infrared spectroscopy(FTIR)scanning,Differential scanning calorimetry(DSC)method,self-assembly curves and Scanning electronic microscopy(SEM).Moreover,the collagen self-assembly membrane materials were prepared by porcine collagen and grass carp scale collagen,and then used to immobilize catalase.These could provide theoretical guidance for expanding the application of collagen.The major conclusions were summarized as following:1.The collagens were extracted by acid-enzyme combination method.SDS-PAGE showed that all the extracted collagens contained two different ? chains(? 1,?2)and one ?chain,they were typical type ? collagen and had not been hydrolyzed into small molecular proteins and peptides.UV and FTIR spectra showed that the extracted collagens had UV absorption peaks in the range of 222-236 nm and owned five infrared absorption bands(amide A,B,?,?,and ?)of type ? collagen.There were some differences in the thermal stability of the six kinds of collagens,especially,mammalian collagens were more stable than aquatic collagens.The maximum peak temperature of pigskin,bovine skin,bovine tendon collagen were 45.37 ??42.65 ??45.73 ? and the denaturation temperature of crucian carp skin,grass carp skin,grass carp scale were 39.05 ??39.75 ??34.49 ?,respectively.2.Turbidity experiments and the degree of fibril formation were employed to study the self-assembly behavior of collagens.The results showed that all the extracted collagens could self-assemble in vitro.The rates of pigskin collagen reached 76%.The self-assembly degrees of collagens from the same species were closed to each other,such as grass carp skin and scale(28%,27.33%),bovine skin and tendon(52.67%,59%).The temperature has a significant influence on the self-assembly of collagen.When the temperature was reduced to 10 ?,no collagens had self-assembly behavior.SEM was used to view the self-assembly structure of collagens.After self-assembled,the clearly fibrous structure was observed in grass carp skin and bovine tendon collagen,each fibril with the periodic stripes D-period exposed completely and staggered to form a fibril web.Two kinds of fish skin collagens also formed a fibrous network structure but the fibril was not exposed completely.The fibers of grass carp skin were slender,while the crucian carp skin were more intertwined.Pigskin and bovine skin collagens formed less fibril with broken phenomenon.3.The collagen self-assembly membranes were prepared by pigskin and grass carp scale collagens.The effects of glutaraldehyde concentration on the sensory properties and mechanical properties of collagen self-assembly membranes were studied.With the increasing of glutaraldehyde concentration,the sensory properties of the two films were changed:their thickness increased,their color varied from light yellow to yellow,and their light transmittances decreased significantly from 51%.In the meanwhile,the increase of glutaraldehyde concentration improved the mechanical properties of the two self-assembly materials,their elongation at break and tensile strength had a certain advance.The infrared spectra indicated that the absorption peaks of the collagen molecules were preserved and the three helix structures were well maintained.Due to the addition of glutaraldehyde,the C=N stretching vibration and the C=O vibration absorption peak were enhanced.The denaturation temperature of pigskin and grass carp skin self-assembly membranes was 80? and 65? respectively,which were higher than that of natural collagen fibers and primitive collagen solution.The results of SEM showed that the increase of glutaraldehyde concentration could damage the fibril structure.4.The catalase was immobilized by crosslinking,adsorption and embedding,respectively.The enzyme activity of embedding-immobilized catalase by grass carp scale collagen self-assembly membrane was the highest,and the operation stability was the best.When the enzyme concentration was 0.5mg/mL,the immobilized enzyme activity of catalase immobilized by the grass carp scale film reached 2596U/g,and the relative activity was more than 50%after using 22 times.The concentration of enzyme and glutaraldehyde both had influence on the immobilization.SEM images showed that the grass carp scale membrane had obvious fibril reticular structure with D-period,and the catalase particles attached to collagen fibrils.The results showed that collagens extracted from pigskin,bovine skin and tendon,grass carp skin and scale,crucian carp skin were type ? collagens.There were some differences in the self-assembly ability and structure of the type ? collagen between different sources.The membrane material was prepared by the self assembly collagen,and it was used to immobilize catalase.The immobilized enzyme showed high activity and stability.It provided a theoretical basis and data reference for the application of collagen based materials.