| In recent years, an important research field has been discovered that collagenpeptide has a great effect on human body, particularly, has characters and functionswhich can be applied into biomedical and biotechnological field. In this thesis,collagen peptide degradated by complex enzyme was studied and systemic andintegrate techniques were set up. It was involved several research aspects: establizatingextraction process of complex enzyme in collagen peptide, and its separation andpurification were studied, The synthesis of metalloprotein peptide and application ofcollagen peptide were also conducted. The sequences of amino acid andthree-dimensional structures were determined. And various functions and performancesof collagen peptide were analyzed and discussed.Hydrolyzing of the scrap leather with complex enzymes. The leather washydrolyzed by complex enzymes, such as acidity protease 3350, basic protease 2833,neutral protease ASI.398, Protamex, and Flavourzyme to extract collagen peptidewithin it. The treatment processes and the influential factors of the enzymes werestudied and the appropriate processing method was presented as follows: pretreatmentby CaO, then enzymatic hydrolyzed by basic protease 2709 and neutral proteaseAS1.398 together, filtrated, concentrated and dried. The results indicated that thecontent of concentrated protein was more than 90%, and the yield was 71.9%. In thework, Abbe refractometer was used to determine the refractive index of solution tocontrol the degree of hydrolyzation. And its feasibility was deduced by the theory, itscorrectness was confirmed by experimentsSynthesization of peptide copper with collagen peptide mentioned above wasexplored. The process conditions were as follows: the pH value of reaction solutionwas at 11, for 20 min at room temperature. The molecular structure of the product wasconfirmed by infrared spectrometry. And the metalloprotein peptide's structure wasdetermined by X-diffraction and nuclear magnetic technology, and three-dimensionalstructure was also characterizedA multi-dimensions technology consisting of the capillary electrophoresis andmass spectrum and IEF-HPLC and database was designed. It made researches simpleand easy. It was used just a little sample (≤100uL) and amortize liquor (5-10mL). Thetechnology is suitable for fast analysis of bio-materials The sequence of collagen peptide was mensurated and its dimensional structurewas also characterized. The spectrograms of peptide, segmental spectrograms ofpeptide, and the formation and structure of amino acid were examined and confirmedby modem analysis methods. The incline and base structure, carton ring and mainframework, second grade structure and fag end, cooperating structure of metalloprotein,crystal structure of collagen peptide, hydration structure and multi-gather structurewere studied accordingly.The fimctions and performances of collagen peptide were discussed. Thethiosulfured was incorporated into collagen peptide, and it was possible to control theactions of dissolved peptide in DBC by disulfide bond. DBC-dissolved peptide provedthe activity of dissolved peptide. It was very stable within 3 months. And its activityafter heat treatment was more stable than free anzyme due to pH value. It was the bestwhen pH value was at 6.3, however, the natural dissolved peptide's was at 9.0.It was found that there was a dashed at 465 nm in photoluminescence intensitywhen concentration of collagen peptide touched to more than 0.5 mg·mL-1; and theintensity could be better with the increase in its concentration. The viscosity of thissystem became higher when the concentration of collagen peptide increased to 0.15mg·mL-1. It was confirmed that collagen peptide has a speciality of collection in itswater solution.Sixteen sorts of objective peptides were separated from other impuritiessuccessfully by high efficiency liquid chromatography , those small peptides withsimilar structures were also analyzed. The trifuoroacetic acid was used as the mobilephase and acid regulator, the effect of different ratio of acetonitrile and water onseparation of peptide was reviewed. Acetonitrile can be separated when its content was20%, and its flow speed was 1.0 mL.min-1. The purity of objective peptide can beranged from 23.78% to 89.9%, and transfer time was in 3.35-27.15 min. It was clearthat high efficiency liquid chromatography was more efficient, for which the purity ofmain peaks were 79.98% and 87.69%, respectively after detecting the standard oftrypsin A chain and trypsin B chain, and its label purity were 80.11%, 89.34%respectively.An efficient way named Tricine-SDS-PAGE to separate small peptides frompeptide was presented in this thesis. It was efficient to separate peptides ranging with 1kD by adjusting the composition of polyacrylamide in colloid, in which the degree ofcross-linking of acrylamide and diacylamide in compact colloid was 5.5% with adding of 38% carbamide. It was much better than that of normal SDS-PAGE and presentTricine-SDS PAGE.Sub-collagen peptide was filtrated and separated kinetically by CE-SDS proteinequipment. The optimal conditions were detected by UV at 220 nm: the size ofcapillary without coated was 36 cm×50um, the voltage of electrophoresis injectionwas 10 kV/10s, the voltage of operation was 15 kV, the temperature of capillary was 20℃. It was found that the data recurred better and in linear in this way. And theconcentration of collagen peptide was 0.125-1.25 mg/mL. DSCE of pigskin collagenpeptide was similar as SDS-PAGE gel stained by Kumaci in numerical and content.The products have been analyzed and detected. The results show that it contains19 kinds of amino acid, the content of protein is more than 90%. The tests ofapplication indicate that collagen peptide with molecular weight ranges from 3000 to4000 Da can play an important role in water collection, oxygenation resistance, and theincrease in skin elasticity.
|
PDF Full Text Request