Studies On High Efficient Preparation Technology And Its Structure Of Type ? Collagen From Scales Of Red Drum Fish(Sciaenops Ocellatus)

This study was financially supported by the scientific research foundation of third institute of oceanography,SOA.(No.2015003),and the cooperate construction project by Xiamen Ocean Research and Development Institute.and the special fund for major science and technology projects in Fujian province(No.2013NZ0003).At present,type ? collagen is the collagen that attracts the most research attention and has the greatest market potential.Type ? collagen from marine fish scales not only satisfy religious customs of Judaism,Islam and Hinduism,and alleviate anxieties of consumers to mammalian collagen that potentially be related to infections such as bovine spongiform encephalopathy(BSE),transmissible spongiform encephalopathy(TSE)and foot-and-mouth disease(FMD).So they can serve as a promising alternative for collagen products from mammals.However,there are still the shortcomings in the study of type ? collagen from marine fish scales as follows:the yield of extraction is generally low;the isolation approach is rather inefficient;the lack of accurate quantitative methods,the scarcity and incompleteness of amino acid sequence;it has not been reported on post-translational modification and function prediction.The purpose of this study is to solve the common problems.The main research contents and the results are as follows.1.Study on Rapid Isolation for PSCPepsin-soluble collagen(PSC)with low antigenicity was extracted from red drum fish scales by pepsin-soluble extraction.Hydrophilic ultrafiltration is introduced to solve the problems about the traditional and time-consuming approach of salting out and dialysis to separate collagen.PSC is isolated rapidly to obtain pepsin-soluble type ? collagen from red drum fish scales.PSC was identified as type ? collagen by SDS-PAGE and its purity was no less than that of rat tail type ? collagen(electrophoretic purity?95%).Hydrophilic ultrafiltration reduces the separation time of collagen from 1 to 3 days to less than 3 hours,and the treatment capacity is increased from less than 1 liter in the traditional dialysis process to 45 liters of collagen solution at a time.The pepsin-soluble type ? collagen of red drum fish scales is PSC in this study.2.Study on Quantification of PSCGel chromatography(GC)and reversed-phase high performance liquid chromatography(RP-HPLC)are used to determine the PSC purity in this study.PSC is determined to be chromatographic pure type ? collagen.RP-HPLC is selected for quantitative analysis and its chromatographic analysis conditions are optimized.According to the rules in Chinese Pharmacopoeia(2015 edition),the peak area normalization method is used to quantitate the PSC purity and to obtain purity data(94.00%±0.01%).The content of PSC(91.27%±0.01%)was calculated by mass balance method.The conversion factor between PSC and hydroxyproline was consequently calculated(11.24±0.26).In the case of insufficient purity of collagen,a conversion factor will contribute to calculation of the collagen content.3.Study on Physicochemical Properties of PSCMass spectrometry,spectroscopy,chromatography,electron microscopy,electrophoresis and other analytical methods were used to characterize the physicochemical properties of PSC.In terms of the thermal stability,the Ubbelohde viscosity method is chosen to determine the denaturation temperature of PSC(27.3 ?).In addition,the exact molecular weight(288.2kDa)of PSC was determined by MALDI-TOF,and the contents of heavy metals in PSC samples was determined by ICP-MS,which ed national standards.4.Study on the Structure of PSCFull-length transcriptome sequencing technique was used to construct the specific CDS protein library of red drum fish scales;In the process of quantitative analysis,the fractions of the main peaks of PSC is collected from RP-HPLC for protein identification.By comparison,not only the amino acid sequence of type ? procollagen a chains are selected.but also PSC was firstly determined according to the distribution of identified peptides.The amino acid sequence belong to type ?collagen by NCBI,Uniprot and KEGG databases.Blast Tree View analysis of type ? procollagen ?1 chain and ?2 chain of red drum fish scales show that they belong to the unknown bony fish protein.The structure of PSC predicted by Phyre2 Protein Fold Recognition Server was similar to that of human fibrous type ? procollagen.There are 96 proline hydroxylation sites and 9 lysine hydroxylation sites in ?1 subunit of PSC,84 proline hydroxylation sites and 5 lysine hydroxylation sites in ?2 subunit are identified.The modification sites of phosphorylation and glycosylation in the amino acid sequence of PSC are predicted by Netphos-3.1,NetGlycate 1.0.and YinOYang1.2 servers.It is proved that the modification of PSC had an effect on its molecular weight,its solubility and its low antigenicity.Meanwhile,the antigenic epitopes of N-terminal peptide and C-terminal peptide of type ? procollagen a chain of red drum fish scales are predicted by ABCpred server.The results showed that the non-helical region peptide chains of type ? procollagen in red drum fish scales had a high proportion of antigenic epitopes,and the N-terminal peptide and C-terminal peptide of collagen are the main antigenic determinants.CDS protein library of red drum fish scales was constructed based on full-length transcriptome sequencing.All proteins in a chains of crude extracted PSC before hydrophilic ultrafiltration and pure PSC after hydrophilic ultrafiltration are identified.The results showed that there are 33 other proteins in a chains of crude extracted PSC before hydrophilic ultrafiltration(26 other proteins in ?1 chain,13 other proteins in ?2 chain,6 other proteins were found in ?1 chain and ?2 chain),while there are only 8 other proteins(of which there are 6 other proteins in ?1 chain,3 other proteins in ?2 chain,1 other protein in ?1 chain and ?2 chain),are found in the ? chain of PSC purified by hydrophilic ultrafiltration.No matter before and after hydrophilic ultrafiltration,there were more other proteins in the ai chain of PSC than in the ?2 chain of PSC.In this study,the amino acid sequences of all other proteins are listed(see Appendix),and by Blast Tree View analysis,there are 25 proteins that have not yet been reported.On the one hand,the results show that hydrophilic ultrafiltration is effective in isolating PSC,which can not only remove impurities outside collagen a chain bands,but also effectively remove the other protein in collagen a-chain bands(removal rate:75.76%);On the other hand,it proved that even if the collagen with electrophoretic purity on SDS-PAGE,there might still be other proteins in the a-chain bands.It is also necessary to further detect and judge the purity of collagen by GC and RP-HPLC with diverse separation mechanisms,which verifies the rationality and necessity of accurate quantification of PSC.More importantly,the results also proved that the combination of full-length transcriptome sequencing and protein identification can obtain not only the amino acid sequence of collagen,but also the amino acid sequence of other proteins.The method has universal applicability and is expected to be a "good prescription" to solve the difficulty of determining the amino acid sequence of proteins without reference genomic.At the same time,eight kinds of other proteins are found in the pure a-chain collagen band of PSC isolated by hydrophilic ultrafiltration,which corresponded to the impurity peaks other than PSC on the RP-HPLC chromatogram.This study not only provides their detailed amino acid sequences,but also confirms their protein structure.Finally,with extensive literature research and amino acid sequence alignment,the domain analysis and efficacy prediction of PSC are completed.In this study,the sites of the stability of the triple helix structure of PSC was analyzed,and the triple helix structure of PSC was further confirmed.It also indicates that PSC has the potential to bind to 24 proteins,polysaccharides and 5 collagen receptors.On this basis,PSC has a specific binding site to integrin.It has been proved that PSC has a large number of binding sites to glycoprotein ?(GP?).PSC also binds directly to two other collagen receptors,the leukocyte-associated immunoglobulin-like receptor(LAIR)and the discoid domain receptor(DDR).It binds to mannose receptor indirectly through fibronectin type ?(Fn?)domain.These direct or indirect binding sites can be found on the amino acid sequence of PSC.Not only that,PSC has clear binding sites to von Willebrand Factor(VWF),pigment epithelium-derived factor(PEDF),platelet reactive protein(TSP),molecular chaperone Hsp47,amyloid precursor protein(APP),osteoclast associated receptor(OSCAR)and heparin.The results show that PSC in this study can participate in and assist different receptors,proteins and polysaccharides to play complex biological functions;at the same time,by comparing the t,ypes of receptors and proteins that can bind to a,chain and a2 chain of PSC,?1 chain is much more than ?2 chain,which is consistent with the situation of ?1 chain and ?2 chain of PSC in the previous section.5.Study on High efficient extraction of PSCThis study adopts the high efficiency extraction technology,low temperature homogenization technology to greatly improve the yield of collagen.PSC prepared by low temperature homogenization technology was determined to reach the electrophoretic purity level.The results of moisturizing and antioxidant activities showed that two PSC not only had excellent moisture absorption ability and moisturizing ability;but also their antioxidant activity was significantly higher than that of type I collagen of rat tail from terrestrial animals and type I collagen of salmon skin from aquatic animals.These results indicate that PSC prepared by low temperature homogenization technology can replace pure PSC in various fields.