Journal of Food Bioactives, ISSN 2637-8752 print, 2637-8779 online
Journal website www.isnff-jfb.com

Original Research

Volume 17, March 2022, pages 34-48

In silico investigation of molecular targets, pharmacokinetics, and biological activities of chicken egg ovalbumin protein hydrolysates

Figures

Figure 1.
Figure 1. Fragments of ovalbumin released by the action of the GI enzymes, chymotrypsin, trypsin, and pepsin.
Figure 2.
Figure 2. eXpression2Kinases Network showing overall interactions of intermediate proteins, kinases and transcription factors with high hypergeometric (−log10) p-value.
Figure 3.
Figure 3. Binding interaction between selected peptides (number) and protein targets (alphabet) as shown in Table 4 and visualized using PyMOL.

Tables

Table 1. Predicted peptides (with minimum of three amino acid residues) of intact ovalbumin (UniProt ID: P01012) with potential biological activity from BIOPEP-UWM
 
Biological activity
Alpha-amylase inhibitorACE inhibitorAntioxidativeOthers
PeptideSequence locationPeptideSequence locationPeptideSequence locationPeptideSequence location
*Anti-inflammatory, **Renin inhibitor, ***Anxiolytic-like peptide, ****Immuno-stimulating peptide, *****Stimulating vasoactive substance release. Bolded = reported in literature. Blue = multiple biological activities. Purple = component of other peptides of the same biological activity.
KLPGF64–68$pVSP$p$p396–398$pVHH21–23$bLPF$b*$b239–241$b
$dEVSGL$d$d 257–261$dILP116–118VHHANEN21–27$bYNL$b**$b300–302$b
QITKPN92–97$pLVL$p$p251–253$pSALAM37–41$bYLG$b***$b43–45$b
$d AEAGVD$d$d 356–361$dLQP166–168$pLWE$p$p190–192$pGLF****222–224
$d EAGVD$d$d 357–361$dLLP253–255AEERYP110–115$dSSS$d *****$d318–320$d
NVLQPS164–169VFK15–17DEDTQAMP197–204
LEPINF134–139LPG65–67$dGAA$d$d 5–7$d
ANENIF24–29FCF11–13KG$pLWE$p188–192
$bQIGLF$b$b220–224$bFFGR$dCVSP$d391–398LFC378–380
HAEIN342–346ERKIKVYL284–291NEN25–27
DHPFLF374–379FGR$dCVSP$d392–398$dSVL$d$d304–306$d
NIFYCP27–32$bYNL$b$b300–302b$
YAEERYPIL109–117$bYLG$b$b43–45$b
$bQIGLF$b220–224
$bLPF$b$b239–241$b
$dAVL$d$d388–390$d
LFR223–225
IFY28–30
$dVVR$d$d57–59$d
LKISQ332–336
INKVVR54–59
VYLPRM289–294
KLP64–66
INKV54–57
$dSQAVH$d$d335–339$d
QAVHA336–340
LT$dSVL$dMA302–308
SLR85–87
$bYNL$b$b300–302$b
$pLVL$pL251–254

 

Table 2. Ovalbumin hydrolysate peptides that were predicted to be biologically active on BIOPEP-UWM
 
Biological activityPeptide
Bolded = peptides with multiple biological activity.
ACE inhibitor$dVY$d, $dPR$d, $dAF$d, IF, $dGL$d, GR, $dSF$d, CF, $dEK$d, $dIL$d, $dAVL$d, $dVVR$d, $dVM$d, $dEF$d
AntioxidativeEL, $dVY$d
CaMPDE or Renin inhibitor$dEF$d, $dSF$d
Glucose uptake stimulating peptide$dVL$d, $dIL$d
Dipeptidyl peptidase IV inhibitor (DPP IV inhibitor)$dEK$d, $dSL$d, $dGL$d, $dAF$d, $dIL$d, IN, $dPF$d, $dSF$d, SW, VH, $dVL$d, VN, $dVM$d, $dVY$d
Dipeptidyl peptidase III inhibitor (DPP-III inhibitor)$dPR$d, $dPF$d, $dVY$d
Hypolipidemic peptide$dEF$d
Regulator of phosphoglycerate kinase activity$dSL$d

 

Table 3. Protein targets of ovalbumin protein hydrolysates predicted from SwissTargetPrediction
 
αHydrolysate peptides (Ligands)% Probability of Predicted Targets
ABCDEFGHIJKLMNOPQR
α indicates active peptide from serial number listed in the supplementary file Table s1, where peptides 1–65 were peptides obtained from enzymatic hydrolyzed protein, and peptide 66-71 were the most active peptides obtained from the unhydrolyzed protein screening. A: HLA class I histocompatibility antigen A-3 (P04439). B: Protein farnesyltransferase (P49354 P49356). C: Calpain 1 (P07384). D: Cathepsin (B and K or D) (P07858 or P07339). E: Complement factor B (P00751). F: Furin (P09958). G: Inhibitor of apoptosis protein 3 (E3 ubiquitin-protein ligase XIAP) (P98170). H: Cyclooxygenase-2 (P35354). I: Angiotensin-converting enzyme (P12821). J: Lipoxin A4 receptor (N-formyl peptide receptor 2) (by homology) (P25090). K: Dipeptidyl peptidase IV (P27487). L: Integrin alpha-V/beta-3 and alpha-5/beta-1 (P06756 P05106 and P05556 P08648). M: Neurokinin 1 receptor (by homology) (P25103). N: Mu/Delta opioid receptors (by homology) (P35372/P41143). O: Beta-secretase 1 (P56817). P: HMG-CoA reductase (P04035). Q: Tyrosyl-tRNA synthetase (P54577). R: Sodium/glucose cotransporter 1 (P13866).
1GSIGAASM25
3CPIAIM3560
4SAL25
5GAK353030
8VVR4030
9PGF403525
11SSL25
13QITK25
17PIL755075
20GGL70
22QTAADQAR50
24GIIR55
26AIVF258530353535/30
29PVQM45
30QIGL502545
42GITDVF3525
44SGISSAESL60
53AVL252540504540
55VY405060
60IL2525
67YLG2540/60
68LPF903050/30
69QIGLF5525
70VSP253545
71LWE50

 

Table 4. Predicted binding energy and dissociation constant of the protein-protein complexes obtained from PRODIGY
 
αHydrolysate peptides (Ligands)Predicted Binding Energy ΔG (kcal.mol−1), Dissociation Constant Kd (10−7M) at 25.0 °C
ABCDEFGHIJKLMNOPQR
α indicates active peptide from serial number listed in the supplementary file Table s1, where peptides 1–65 were peptides obtained from enzymatic hydrolyzed protein, and peptide 66–71 were the most active peptides obtained from the unhydrolyzed screening. A: HLA class I histocompatibility antigen A-3 (1AKJ). B: Protein farnesyltransferase (2H6I). C: Calpain 1 (AF-P07384-F1-model_v2). D: Cathepsin (B and K) (2PBH). E: Complement factor B (2OK5). F: Furin (6HLD). G: Inhibitor of apoptosis protein 3 (E3 ubiquitin-protein ligase XIAP) (AF-P98170-F1-model_v2). H: Cyclooxygenase-2 (5KIR). I: Angiotensin-converting enzyme (4C2N). J: Lipoxin A4 receptor (human formyl peptide receptor 2) (by homology) (6LW5). K: Dipeptidyl peptidase IV (3Q8W). L: Integrin alpha-V/beta-3 and alpha-5/beta-1 (4O02). M: Neurokinin 1 receptor (by homology) (6HLO). N: Mu/Delta opioid receptors (by homology) (AF-P35372-F1-model_v2). O: Beta-secretase 1 (2OHM). P: HMG-CoA reductase (AF-P04035-F1-model_v2). Q: Tyrosyl-tRNA synthetase (4QBT). R: Sodium/glucose cotransporter 1 (AF-P13866-F1-model_v2). AF represent Alphafold monomer v2.0 prediction obtained from UniProt structure section for each of the selected proteins, while structures of other proteins were obtained from RCSB protein databank.
1GSIGAASM−8.5, 6.2
3CPIAIM−8.3, 8.2−9.6, 0.93
4SAL−7.9, 17.0
5GAK−7.5, 29.0−6.7, 120.0−7.7, 21.0
8VVR−7.2, 56.0−7.9, 17.0
9PGF−7.3, 42.0−6.5, 180.0−8.9, 2.9
11SSL−8.6, 5.0
13QITK−5.9, 450.0
17PIL−7.3, 42.0−7.7, 21.0−8.4, 7.2
20GGL−8.3, 8.2
22QTAADQAR−8.9, 2.9
24GIIR−8.9, 2.9
26AIVF−8.8, 3.4−8.0, 13.0−8.1, 12.0−7.8, 19.0−5.6, 820.0−7.6, 27.0
29PVQM−8.3, 8.2
30QIGL−7.5, 29.0−8.2, 9.3−8.5, 5.4
42GITDVF−8.4, 7.2−5.9, 460.0
44SGISSAESL−9.5, 1.1
53AVL−6.8, 100.0−4.8, 2800.0−7.3, 45.0−8.0, 13.0−8.3, 8.2−8.9, 2.8
55VY−5.1, 1800.0−4.1, 9700.0−5.3, 1200.0
60IL−4.8, 2800.0−7.9, 17.0
67YLG−7.6, 27.0−5.7, 670.0
68LPF−7.2, 53.0−7.5, 29.0−6.2, 280.0
69QIGLF−8.0, 13.0−8.0, 13.0
70VSP−8.0, 13.0−5.2, 1500.0−6.7, 120.0
71LWE−8.0, 13.0

 

Table 5. Predicted pharmacokinetics properties of ovalbumin protein hydrolysates
 
αHydrolysate peptides (Ligands)Predicted ADME Parameter
MWMRTPSA (Å2)Log PESOL Log SESOL ClassGIABBBP-gpCYPs InhibitorLog Kp (cm/s)BSSA
α indicates active peptide from serial number listed in the supplementary file Table s1, where peptides 1–65 were peptides obtained from enzymatic hydrolyzed protein, and peptide 66–71 were the most active peptides obtained from the unhydrolyzed screening. Legend: Physicochemical properties: Molecular weight (MW), Molar Refractivity (MR), Total polar surface area (TPSA). Lipophilicity: Consensus Log P. Water solubility: ESOL Log S, ESOL Class. Pharmacokinetics: Gastrointestinal absorption (GIA), Blood-brain barrier (BBB), P-glycoprotein (P-gp) substrate, Inhibition of Cytochrome P450 (CYPs) type CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4, Skin permeation (Log Kp). Drug-likeness: Bioavailability Score (BS), Medicinal chemistry: Synthetic accessibility (SA).
1GSIGAASM692.78167.31332.78−3.051.38Highly solubleLowNoYesNo−14.580.176.10
3CPIAIM646.86173.61264.130.4−0.78Very solubleLowNoNoNo−11.990.176.06
4SAL289.3371.44141.75−1.391.14Highly solubleLowNoNoNo−10.440.553.20
5GAK274.3268.18147.54−1.541.79Highly solubleLowNoNoNo−10.910.552.89
8VVR372.4698.61183.42−1.141.57Highly solubleLowNoYesNo−11.720.553.92
9PGF319.3687.05107.53−0.220.29Highly solubleHighNoNoNo−10.180.552.85
11SSL305.3372.60161.98−21.97Highly solubleLowNoNoNo−11.510.553.24
13QITK488.58123.32239.96−1.651.67Highly solubleLowNoYesNo−12.910.174.90
17PIL341.4596.21107.530.59−0.2Very solubleHighNoYesNo−9.550.553.69
20GGL245.2860.67121.52−1.11.32Highly solubleHighNoNoNo−10.150.552.31
22QTAADQAR859.88204.08472.63−5.643.13Highly solubleLowNoYesNo−18.220.177.25
24GIIR457.57120.84212.52−0.510.05Highly solubleLowNoYesNo−10.750.174.69
26AIVF448.56121.8150.621.2−1.4Very solubleLowNoYesNo−9.460.554.22
29PVQM473.59124.13205.02−1.31.23Highly solubleLowNoNoNo−12.440.174.66
30QIGL429.51109.84193.71−0.671.07Highly solubleLowNoYesNo−11.680.174.14
42GITDVF650.72164.39266.35−0.62−1.07Very solubleLowNoYesNo−11.750.115.69
44SGISSAESL849.88200.46414.34−4.721.99Highly solubleLowNoYesNo−16.810.117.02
53AVL301.3879.89121.52−0.080.72Highly solubleHighNoNoNo−10.130.553.25
55VY280.3274.56112.650.010.25Highly solubleHighNoNoNo−9.800.552.64
60IL244.3367.2892.420.490.25Highly solubleHighNoNoNo−9.000.552.87
67YLG351.4091.98141.75−0.020.14Highly solubleLowNoNoNo−10.260.553.04
68LPF375.46106.28112.730.85−0.86Very solubleHighNoYesNo−9.510.553.52
69QIGLF576.68151.75222.810.16−0.55Very solubleLowNoYesNo−11.530.175.00
70VSP301.3478.14132.96−1.411.53Highly solubleLowNoYesNo−11.200.553.27
71LWE446.5118.01174.610.41−0.95Very solubleLowNoNoNo−10.090.114.06

 

Table 6. Predicted physicochemical properties of ovalbumin protein hydrolysates
 
αHydrolysate peptides (Ligands)Predicted Physicochemical Parameter
ToxicityHemolytic (SVM score)pIHydrophobicityHydropathicityHydrophilicityCharge
α indicates active peptide from serial number listed in the supplementary file Table s1, where peptides 1–65 were peptides obtained from enzymatic hydrolyzed protein, and peptide 66–71 were the most active peptides obtained from the unhydrolyzed screening. ToxinPred webserver based on support vector machine (SVM) score: positive (YES) or negative (NO). Hemolytic prediction based on SVM score.
1GSIGAASMNo0.495.880.160.95−0.440.00
3CPIAIMNo0.495.850.322.27−1.070.00
4SALNo0.495.880.171.60−0.670.00
5GAKNo0.499.11−0.23−0.830.831.00
8VVRNo0.4910.11−0.231.300.001.00
9PGFNo0.495.880.230.27−0.830.00
11SSLNo0.495.880.000.73−0.400.00
13QITKNo0.499.11−0.31−0.900.251.00
17PILNo0.495.880.402.23−1.200.00
20GGLNo0.495.880.281.00−0.600.00
22QTAADQARNo0.496.19−0.41−1.290.560.00
24GIIRNo0.4910.11−0.031.02−0.151.00
26AIVFNo0.495.880.533.33−1.570.00
29PVQMNo0.495.880.010.25−0.650.00
30QIGLNo0.495.880.181.10−0.850.00
42GITDVFNo0.493.800.191.15−0.53−1.00
44SGISSAESLNo0.494.000.000.330.01−1.00
53AVLNo0.495.880.443.27−1.270.00
55VYNo0.495.880.281.45−1.900.00
60ILNo0.495.880.634.15−1.800.00
67YLGNo0.495.880.240.70−1.370.00
68LPFNo0.495.880.361.67−1.430.00
69QIGLFNo0.495.880.271.44−1.180.00
70VSPNo0.495.880.070.60−0.400.00
71LWENo0.494.000.09−0.20−0.73−1.00

 

Table 7. Predicted therapeutic potential of the peptides
 
αHydrolysate peptides (Ligands)Antimicrobial*Antioxidant**Anticancer***Anti-inflammatory****
AntibacterialAntiviralAntifungal
α indicates active peptide from serial number listed in the supplementary file Table s1, where peptides 1–65 were peptides obtained from enzymatic hydrolyzed protein, and peptide 66–71 were the most active peptides obtained from the unhydrolyzed screening. * = 0.5 Probability cut-off. ** = 0.5 Probability cut-off. *** = SVM score: positive (YES) or negative (NO).
1GSIGAASMYESNOYESNONOLOW
3CPIAIMYESYESYESNOYESLOW
4SALNONONONOYESNO
5GAKYESYESYESNONONO
8VVRYESYESNONOYESNO
9PGFYESYESYESNOYESNO
11SSLYESNONONOYESNO
13QITKYESYESNONOYESNO
17PILYESYESYESNOYESNO
20GGLYESYESYESNONONO
22QTAADQARNONONONOYESLOW
24GIIRYESYESYESNONOMEDIUM
26AIVFYESNONONOYESLOW
29PVQMYESYESNONOYESNO
30QIGLYESYESYESNOYESLOW
42GITDVFNONONONONONO
44SGISSAESLNONONONOYESLOW
53AVLYESYESNONOYESNO
55VYYESYESNONOYESNO
60ILYESYESYESNOYESNO
67YLGYESNONONOYESNO
68LPFYESYESYESNOYESNO
69QIGLFYESNOYESNOYESMEDIUM
70VSPNONONONOYESNO
71LWEYESYESNONOYESNO
**** ConfidenceScore rangeSensitivitySpecificity
α indicates active peptide from serial number listed in the supplementary file Table s1, where peptides 1–65 were peptides obtained from enzymatic hydrolyzed protein, and peptide 66–71 were the most active peptides obtained from the unhydrolyzed screening. * = 0.5 Probability cut-off. ** = 0.5 Probability cut-off. *** = SVM score: positive (YES) or negative (NO).
HighScore ≥ 0.46863.22%90.30%
Medium0.468 > Score ≥ 0.38871.90%80.11%
Low0.388 >Score ≤ 0.34278.39%70.87%