The Discovery Of Thermostable Antioxidant Peptides In Musca Domestica And Bioinformatics Analysis

Antioxidant peptides become the research hotspot with small molecular weight,.easy to absorb,remarkable effect.By collating relevant research data at home and abroad,it showed that many antioxidant peptides were not widely used,mainly limited to its stability.In this study,a kind of thermostable antioxidant peptides was found and proved stability in a variety of applications in the environment.The basic properties and spatial structure characteristics of the peptides were analyzed and were cleared the application conditions.The thermostable antioxidant peptides were proved in housefly maggot bots for the first time,and the action mechanism about stability and antioxidant at primary structure and advanced structure also illuminated.This result will be a certain basis of the application of high temperature resistant antioxidant peptides for the future research.The main research results are as follows:Using SPSS21 software analyzed correlation between three methods（NBT,DPPH,FRAP）of antioxidant,the results showed that there were positive correlation and negative correlation between them,in order to comprehensive evaluation of protein oxidation resistance determination by three methods together.Dealed with50 ℃,80 ℃ and 100 ℃ water bath heating for 25 min,the results of antioxidant activity and protein sds-page electrophoresis showed that 100 ℃ for 10 min can remove impurities efficiently,keep relatively stable oxidation resistance.Besides the correlation between antioxidant activity and protein showed that the antioxidants was proteins,and NBT staining determine the antioxidant peptide molecular weight was about 10 KD.Through DEAE cellulose column chromatography to separate and purify protein,the results showed the optimal separation condition : eluent for the A liquid(0.02 mol.L^-1 pH = 7.0 phosphate buffer)and B liquid(0.02 mol.L^-1 pH = 7.0 phosphate buffer +5% Nacl)mixture,A liquid from 100% to 0,B liquid by 0 to 100%,mixed different concentration gradient for12 min,flow rate of 3 ml/min,gradient elution for12 min.The separation condition of hydroxyapatite chromatography was: eluent for the A liquid(0.02 mo.L^-1 pH = 7.0 phosphate buffer)and B liquid(0.2 mol.L^-1 pH= 7.0 sodium phosphate buffer)mixture,A liquid from 100% to 0,B liquid by 0 to100%,mixed different concentration gradient for12 min,flow rate of 3 ml/min,gradient elution for12 min.After two kinds of chromatography,the result of protein sds-page showed that the protein of 20 KD molecular weight was separated and purifies about 10 KD.In order to comprehensive evaluation of antioxidant peptide oxidation resistance in the high temperature,different pH,ultraviolet irradiation and digestive enzyme processing conditions,the methods of NBT,DPPH and FRAP were used.The results showed that the antioxidant peptides remained higher antioxidant activity at 100 ℃and 120 ℃water bath treatment for a long time;antioxidant peptides had good stability in acid condition,the optimum pH= 2 for reducing power,the optimal pH =3for removing DPPH free radical;the superoxide anion free radical clearance remained stable,DPPH free radicals clearance remained at around 84%,and reducing power descended 46% under the ultraviolet for a long time;the superoxide anion free radical clearance reduced significantly treated pepsin digestion but not chymotrypsin digestion,DPPH free radical clearance keeped stable,reducing power rose significantly.8 kinds of thermostable antioxidant peptides were screened out with protein spectrum: Animal heme peroxidase、Hemolymph juvenile hormone binding protein、Redoxin 、 Glutathione peroxidase and4 kind of Cu-Zn SOD.Their physical and chemical properties,the hydrophobicity,transmembrane domain structure,signal peptide,senior biological information were analyzed.Results showed that the protein generally contained a higher concentration of glycine,alanine and valine,low tryptophan,cysteine,methionine and tyrosine;protein stability coefficient were low,containing specific domain of conservative structure;they were hydrophilic proteins,containing peptide binding sites of glutamic acid,not containing transmembrane region;the number of alpha helix was less than beta sheet,containing a lot of alanine but cysteine near the peptide chain N terminal or C distribution;with a short chain or without at C and N terminal,but the proteins generally had long random-coils.