| Peroxidase?POD?is a metalloenzyme with iron porphyrin as a prosthetic group,widely distributed in microorganisms,plants and animals.There are many types of peroxidases according to their location,peroxidase can be divided into two superfamily of animal origin,plant and microorganism origin according to their location.Different types of peroxidases participate in different biochemical reactions in the creatures.The current researches concerning peroxidase mainly focuses on the plant's in vivo effects and in vitro applications.The in vivo effects involve in plant growth,development,environment stress,photosynthesis,respiration and enzymatic browning.Peroxidase plays an important role in sewage treatment,biosensor preparation,life science,medical clinical diagnosis and industrial brewingPeroxidase is a key enzyme regulating growth and development in plants,which can catalyze phenolic compounds into quinones with H2O2 as an electron donor by oxidation reactions.Quinones then form brown or black spots on the surface of sweet potato through polymerization.Peroxidases immediately contact with phenolic compounds when get mechanically injured,and gave rise to browning phenomenon of plants.Not only browning exerted effects to nutrition value,taste,appearance and cause waste of sweet potatoes,but seriously hinder the development of sweet potatoes industry.Presently,most study concentrated on the aspect of control of browning and reduced its negative influences,while potential molecular mechanism of its occurrence of enzymatic browning and inhibition by inhibitors is unclear.Some commercially synthetic inhibitors on the market such as kojic acid,sodium metabisulfite caused health risks.Therefore,the discovery and screening of non-toxic and highly efficient new peroxidase inhibitors are of great significance for inhibiting browning of sweet potato and reducing losses.The inhibitory effect of using inhibitors to control enzymatic browning is obvious,so it has attracted widespread attention.In this study,two peroxidase isozymes were separated,purified and identified by using chromatography and mass spectrometry,and bioinformatics analysis were performed.Combined with ultraviolet spectrophotometer,fluorescence spectroscopy?FT-IR?,homology modeling,molecular docking and other technologies have studied catalytic binding sites,potential inhibitor mechanism and structure-activity relationship between peroxidase and synthetic inhibitors?ascorbic acid,sodium metabisulfite,reduced glutathione?,natural inhibitors?luteolin,phytic acid?.The main results in this article are summarized as follows:1.Bioinformatics analysis found that there are currently 23 genes encoding 23peroxidases in sweet potato.Sequence alignment revealed that there were conserved regions in the protein encoded by the sweet potato peroxidase gene.Phylogenetic tree analysis showed sweet potatoes were closer to horseradish,Arabidopsis,and cucumber.2.Two peroxidase isozymes,SPOD1 and SPOD2,were successfully obtained from‘Zhongshu 1'by chromatography.Enzyme activity,purification-fold,activity recovery of SPOD1 were 1230527.7 U mg-1,171.74,8.38%,respectively;Enzyme activity,purification-fold,activity recovery of SPOD2 were 813053.57 U mg-1,60.01,11.12%,respectively.SDS-PAGE showed molecular weight of SPOD1,SPOD2 were 37.2 kDa and34.5kDa,respectively.Total molecular weight were 37.0 kDa and 35.1 kDa determined by standard gel filtration chromatography,indicating the two isozymes were monomer.Native-Page revealed the difference in charge and molecular weight due to different positions on gel,indicating SPOD1,SPOD2 were isozymes.3.The optimal pH of SPOD1 and SPOD2 were 4.8,6.4,and remained stable in the pH 4?6.The optimum temperature were 65?and 45?.The activity of SPOD1remained stable in the wide temperature range of 25?55?;the activity of SPOD1remained stable in the range of 25?65?.Metal ions such as Mg2+,Zn2+,Ba2+,Fe3+were SPOD1and SPOD2 activators.Mn2+,Cu 2+showed double role in activation at low concentrations and inhibitory effects at high concentrations.Ca2+had no obvious effect on isozyme activity.Ascorbic acid,SDS,oxalic acid,KSCN and other compounds strongly inhibited the activity of SPOD1 and SPOD2.EDTA showed activation under low concentration conditions as a chelating agent.Km values of SPOD1 and SPOD2were measured by fixing different concentrations of guaiacol to 32.31,19.75 mmol L-1,respectively.The type of the double substrate enzymatic kinetic reaction of the'Zhongshu 1'peroxidase isozyme belonged to be Ping-Pong reaction.4.SPOD1 and SPOD2 were identified by nESI-LC-MS/MS,and the corresponding sweet potato peroxidase were K9PI1 and Q5JBR5 in databank,and the converge rate were 24.96%and 4.89%,respectively.SPOD1 and SPOD2 used the peroxidase 4USC and 1QO4 as templates to obtain ideal models.The molecular docking results of isozyme and guaiacol showed that residues Ala191,Arg61 in SPOD1 were conjugated with guaiacol to form hydrophobic interaction.The residue Pro163 in SPOD2 formed hydrogen bonds with guaiacol and the residue Phe64 formed a hydrophobic interaction with guaiacol.Ala191,Arg61,Pro163,Phe64 were key amino acids that inhibited enzymatic browning.Hydrogen bonding and hydrophobic interaction were important driving forces in catalytic process of peroxidase.5.Ascorbic acid?sodium metabisulfite?reduced glutathione exerted inhibitory effects to SPOD1 and SPOD2.Ascorbic acid was a competitive inhibitor with IC50(1.97×10-4;2.3×10-4 mmol L-1)and Ki(4.95×10-5;5.0×10-5 mol L-1).Sodium metabisulfite influenced the activity of isozymes in a non-competitive inhibition manner with IC50(3.52×10-4;3.11×10-4 mmol L-1)?Kis(4.46×10-5;9.9×10-5mol L-1).Reduced glutathione inactivated isozymes as anti-competitive inhibitors,its IC50 were6.37×10-3,3.35×10-3 mol L-1,respectively,and Kis were 5.37×10-4;99.01×10-44 mol L-11 mol L-1 mol L-1,respectively.Ascorbic acid formed hydrogen bonds with residues Leu188?Ser189 in SPOD1,and Ala192 amino acid residues in SPOD2,respectively.Sodium metabisulfite formed hydrogen bonds with His198,and forms hydrophobic interactions with Arg61;it formed hydrogen bonds with His65 and Pro163 residues in SPOD2,and formed electrostatic interaction with Arg61.Reduced glutathione interacted with residues Val196,Arg61,Ser97,His198,Phe64 in a hydrogen bonds manner,and His163,Arg61,Ala70,Asn72,Ser269 residues in SPOD2 as well.6.Luteolin and phytic acid were potential inhibitors of enzymatic browning.IC50values of luteolin as non-competitive inhibitor on SPOD1 and SPOD2 were 9.98×10-5mol L-1,10.02×10-5 mol L-1.The IC50 values of phytic acid as a competitive inhibitor for SPOD1 and SPOD2 were 1.18×10-6,1.35×10-6 mol L-1.Inhibition order:Phytic acid>luteolin.7.Luteolin and phytic acid quenched the endogenous fluorescence of SPOD1?SPOD2 in a static manner and spontaneously combined a single site in SPOD?SPOD2.The binding distance r<8 nm,0.5 R0<r<1.50 R0,indicating that non-radiative energy transfer occurred in the interaction of phytic acid and luteolin with SPOD1 and SPOD2.Hydrophobic interaction and hydrogen bonds were the main driving forces for the combination of phytic acid and luteolin with SPOD1 and SPOD2.Phytic acid formed hydrogen bonds with residues Gly167,Pro34,Asn72,Cys168 in SPOD1;Asp245,Thr248,Asp250,Arg197,Pro132,Pro38 in SPOD2 as well.Luteolin interacted with residues Pro132,Arg172,Pro132,Pro38 hydrogen bonds in SPOD1;Cys168,Pro38,Ala134 and benzene ring of luteolin had a hydrophobic interaction;Arg197 in SPOD2was connected to luteolin through hydrogen bonds;His193,Arg61,Ala192,Ile60,Phe64had hydrophobic interaction with luteolin.Phytic acid and luteolin inserted into the active sites of SPOD1 and SPOD2 and interacted with the corresponding amino acid residues to formed hydrogen bonds and hydrophobic interactions,and prevented the substrates from entering the catalytic active channel and sharping the affinity with SPOD?SPOD2,eventually leaded to a decrease in the catalytic efficiency of SPOD1 and SPOD28.The combination of luteolin and phytic acid with SPOD1 and SPOD2 reduced the fluorescence intensity of tryptophan and tyrosine,and the microenvironment of tryptophan changes.The interaction of inhibitors with SPOD1 and SPOD2 increased the content of?-helix and random coil in the secondary structure;the decrease of?-sheet and?-turn content directly affected the activity and catalytic efficiency of the enzyme. |
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