Regarding KMUTT’S PhD candidate’sresearch,thein silico digestion results of A. platensisC1 with 15 enzymes led to a protease selection for validation. The bioactive peptide classifi. The result of discovered peptides withantihypertensive activity was selected as a target. She validated the result in thelaboratory by protein hydrolysis with thermolysin. The hydrolysate was further analyzed by LC-MS/MS.
Several potential bioactive peptide candidateswere obtained and tested with ACE inhibition assay.Interestingly, a novel antihypertensive peptide, AHP, have been discovered [61].
In this study, to determine any common characteristics of the ExactlyAHP with the novel one,wehave analyzed 460 predicted antihypertensive peptidesfrom in silico digestionby Clustal Omega (Appendix B). Table 3.2shows 10 common residues, i.e. G, P, Y, L, T, W, R, F, A, and V. The result shows that Group P, G, Y, L are the big groups and contain small sub-groups. Thenovel peptide, AHP, was in the group Y,sub-group YY (Fig 3.8).
31
The Novel AHP, ExactlyAHP_284 and ExactlyAHP_460 belong to cluter group ―xYYx‖. The Novel AHP has the bioactivity simallar to ExactlyAHP_284 and ExactlyAHP_460 as antihypertensive. Besides, Novel AHP have the bioactivity different such as: antioxidant activity, antimicrobialactivity, CaMPDE inhibitoractivity, Hypotensive activity, and Neuropeptide activity(Table 3.3).
These peptides have the bioactivity that found fromA. platensisC1 protein.
Table 3.2 Classification of common amino acid of candidate antihypertensive peptides, discovered fromA. platensisC1.
Group Common Number of peptides
G
xGx 68
xYGx 5
xGLx 2
xGIx 2
xGYx 3
xKGx 2
xHGx 2
xGHx 3
xGVx 4
xFGx 4
xGGx 3
xQGx 2
xGFx 2
xGTx 4
xDGx 2
xAGx 7
xG 13
Gx 8
P
xPx 174
xYPx 15
xPSx 2
xPYx 3
xPAx 3
xHPx 2
xIYPx 3
xPRx 3
xIPx 9
xALPPx 2
xAPx 14
xLKPx 3
xLNPx 2
xPLx 5
xLPx 19
xRPx 5
xMPx 2
32 Group Common Number of peptides
xQPx 4
xKPx 5
xVPx 3
xPPx 6
xPQx 2
xPGx 2
xHPx 3
xFPx 4
xGPx 4
xPRx 3
xNPx 6
xEPx 4
xP 22
Px 5
xPx 9
Y
xYx 65
xVYx 7
xYNx 3
xKYx 6
xIYx 7
xFYx 4
xRYx 5
xYLx 2
xYKx 2
xYAx 2
xEYx 2
xSYx 3
xYSx 3
xAYx 3
xY 7
Yx 6
xYYx 3
L
xLx 47
xLSx 2
xLAx 4
xDLx 3
xRLx 2
xFLx 4
xKLx 3
xLVx 4
xLNx 3
xWLx 2
xHLx 3
xLQx 6
xLFx 3
Lx 6
xL 2
T xTx 5
33 Group Common Number of peptides
W xWx 16
R xRx 6
F xFx 23
A xAx 23
V xVx 10
Other 23
Fig 3.8 Common amino acid sequence of candidate antihypertensive peptideswith Novel AHP
32
Table 3.3Characteristics of Novel AHP based on ExactAHP_284 and ExactAHP_460 sequences.
Name Sequence
Amino acid
sequence Bioactive peptide Name of protein
Novel AHP IRDLDYY
Antihypertensive activity, Antimicrobial activity, Antioxidant activity, Bitter taste, CaMPDE inhibitor activity, Hypotensive activity, Ion flow regulating,
Neuropeptide activity
allophycocyanin beta subunit
ExactAHP_284 MYY Antihypertensive activity, Antioxidant activity
hypothetical protein SPLC1_S206760; hypothetical protein SPLC1_S206770; peptidase M10A and M12B matrixin and adamalysin; diguanylate cyclase/phosphodiesterase; hypothetical protein SPLC1_S103190; PilT protein domain protein; pyruvate dehydrogenase (acetyl-transferring) E1 component alphasubunit;
ErfK/YbiS/YcfS/YnhG family protein; hypothetical protein SPLC1_S630380; hypothetical protein SPLC1_S031960;
glycogen debranching enzyme GlgX; amino acid carrier protein
ExactAHP_460 YY Antihypertensive activity hypothetical protein SPLC1_S501250; hypothetical protein
33
Name Sequence
Amino acid
sequence Bioactive peptide Name of protein
SPLC1_S010200; glycosyl transferase family 2;
methyltransferase type 12; hypothetical protein SPLC1_S011220;
hypothetical protein SPLC1_S011400; RNA polymerase sigma- 24 subunit ECF subfamily; putative type III restriction- modification system Res subunit; SH3 type 3 domain protein;
hemolysin-type calcium-binding region; tetratricopeptide TPR_2;
serine/threonine protein kinase; magnesium chelatase H subunit;
putative sulfotransferase protein; tetratricopeptide TPR_2 repeat protein; hypothetical protein SPLC1_S041230; hypothetical protein SPLC1_S041240; hypothetical protein SPLC1_S050010;
hypothetical protein SPLC1_S050750; methyltransferase;
hypothetical protein SPLC1_S052040; hypothetical protein SPLC1_S060700; aldo/keto reductase; DNA or RNA helicase of superfamily II; diguanylate cyclase with PAS/PAC sensor; DNA methylase N-4/N-6 domain protein; TRAP dicarboxylate transporter- DctP subunit; glycosyl transferase group 1;
hypothetical protein SPLC1_S0825
34
According Table 3.4-3.5, Fig 3.9 and Appendix C, the resultsdemonstrate Aliphatic functional group occupies 675 aminoacids(49,63%) in the total amino acid of only Exactly_AHP from A. platensisC1 including A,G,I,L,P,V. It is the most in the 7 functional groupsthat found in antihypertensive peptide from A. platensisC1.
Table 3.4Amount and percentage of 7functional amino acid groupsof
antihypertensive peptides fromA. platensisC1.
Functional group Total Percent(%)
Aliphatic 675 49,63
Aromatic 314 23,09
Acidic 56 4,12
Basic 168 12,35
Hydroxylic 70 5,15
Sulphur-containing 27 1,99
Amidic 50 3,68
35 49.63
23.09 4.12
12.35
5.15 1.99 3.68 0
Aliphatic Aromatic Acidic
Basic Hydroxylic Sulphur-containing
Amidic
Fig3.9Percentage of 7 functional groups in the amino acid antihypertensive peptide sequences of A. platensisC1.
In addition, Table 3.5 shows amino acid P occupies 184 amino acids and it wasfound to be the most in the 19 amino acids in Exactly_AHP ofA. platensisC1.
However, amino acid N was not found.
Table 3.5Number of amino acidsfound in Exactly_AHP of A. platensisC1.
A G I L P V F W Y D
114 100 80 139 184 48 139 48 127 18
E R H K S T C M N Q
38 62 36 70 42 18 3 24 0 50
Table 3.6Functional amino acid pattern of Exactly_AHP of A. platensisC1.
No Pattern Number
1 Aliphatic 63
2 Aliphatic and Hydroxylic 21
3 Aliphatic and Basic and Sulphur-containing 2
4 Aliphatic and Acidic 23
5 Aliphatic and Aromatic 84
6 Aliphatic and Basic 57
7 Aliphatic and Aromatic and Basic and Hydroxylic 1
36
No Pattern Number
8 Aliphatic and Aromatic and Basic 36
9 Aliphatic and Sulphur-containing 6
10 Aliphatic and Hydroxylic and Sulphur-containing and Amidic 1
11 Aliphatic and Aromatic and Hydroxylic 21
12 Aliphatic and Aromatic and Amidic 6
13 Aromatic and Sulphur-containing 7
14 Aromatic and Acidic 5
15 Aliphatic and Aromatic and Acidic 12
16 Acidic and Basic 4
17 Acidic and Sulphur-containing 2
18 Aliphatic and Acidic and Basic 2
19 Aliphatic and Aromatic and Acidic and Basic 4
20 Aromatic 4
21 Aromatic and Amidic 8
22 Aromatic and Basic 17
23 Aromatic and Hydroxylic and Amidic 2
24 Aliphatic and Amidic 23
25 Aliphatic and Aromatic and Hydroxylic and Amidic 5
26 Basic 3
27 Basic and Amidic 3
28 Aliphatic and Hydroxylic and Amidic 3
29 Basic and Sulphur-containing and Amidic 1
30 Aliphatic and Basic and Hydroxylic 4
31 Aliphatic and Aromatic and Basic and Amidic 4
32 Aliphatic and Basic and Sulphur-containing and Amidic 1 33 Aliphatic and Aromatic and Sulphur-containing 2
34 Aliphatic and Acidic and Sulphur-containing 1
35 Aliphatic and Aromatic and Acidic and Sulphur-containing 1
36 Aromatic and Basic and Sulphur-containing 2
37 Aliphatic and Sulphur-containing and Amidic 1
38 Aliphatic and Basic and Amidic 3
39 Basic and Hydroxylic 1
40 Aromatic and Basic and Amidic 2
41 Aliphatic and Acidic and Hydroxylic 1
42 Aromatic and Hydroxylic 7
43 Aliphatic and Basic and Hydroxylic and Amidic 1
44 Acidic and Hydroxylic 1
45 Hydroxylic and Amidic 2