Bioactive peptides discovery from spirulina classification of common characteristics of antihypertensive peptides

Bioactive peptides discovery from spirulina classification of common characteristics of antihypertensive peptides Bioactive peptides discovery from spirulina classification of common characteristics of antihypertensive peptides Bioactive peptides discovery from spirulina classification of common characteristics of antihypertensive peptides Bioactive peptides discovery from spirulina classification of common characteristics of antihypertensive peptides

THAI NGUYEN UNIVERSITY

UNIVERSITY OF AGRICULTURE AND FORESTRY

LUONG TAI CHIEN

Topic title:

BIOACTIVE PEPTIDES DISCOVERY

FROM SPIRULINA: CLASSIFICATION OF COMMON

CHARACTERISTICS OF ANTIHYPERTENSIVE PEPTIDES

THAI NGUYEN UNIVERSITY

UNIVERSITY OF AGRICULTURE AND FORESTRY

LUONG TAI CHIEN

Topic title:

BIOACTIVE PEPTIDES DISCOVERY

FROM SPIRULINA: CLASSIFICATION OF COMMON

CHARACTERISTICS OF ANTIHYPERTENSIVE PEPTIDES

Supervisors :Asst Prof Dr Marasri Ruengjitchatchawalya

Dr Duong Van Cuong

DOCUMENTATION PAGE WITH ABSTRACT Thai Nguyen University of Agriculture and Forestry

Thesis title Bioactive peptides discovery from Spirulina: Classification

of common characteristics of antihypertensive peptides

Dr Duong Van Cuong

Abstract:

Arthrospira (Spirulina)platensisis a spiral filamentous cyanobacteriumcontaining high protein content about 55-70%as dry weight The alga has been used as human food- and animal feed supplements.Attempt to discover

bioactive peptides from A platensis, Algal Biotechnology research group at King

Mongkut’s University of Technology Thonburi (KMUTT), Thailand,have

investigated an in-silico digestion of the algal proteome and classified the obtained

peptides regarding their bioactivities

In this report, hands-on experience about the algal, A platensisC1, cultivation;

cell harvesting, as well as protein extraction/ separation and a bioactivity assay, hypertensive activity (AH) of some obtained peptides has been described In addition, using a computational tool, Clustal Omega, to determine common characteristics of the putative anti-hypertensive peptides showed that 10 common amino acidgroups were classified, including 4 major groups of G, P, Y and L The reference Novel AH peptidewas in thegroup Y with the common amino acids―YY‖ Besides, these peptides were mostly contained both aliphatic and aromatic amino acid (A, G, I, L,

anti-P, V, F, W, Y) and also hydrophobic amino acid residue (A, I, L, F, V, anti-P, G)

Keywords Spirulina (Arthrospira) platensis C1, Bioactive peptides,

common characteristics

Number of pages 37

formaking experiments look amusing Big thanks also go to every people on Algal Biotechnology Laboratory (AGB research group) and Bioinformatics and Systems Biology (BIF) Program at KMUTT for helping me fit in and feel welcome from the moment and for the unlimited patience to explain me every doubt I had during my internship.I would also like to acknowledge my teachers

at TUAF, Dr Duong Van Cuong, MSc Trinh Thi Chung, Dr Nguyen Xuan Vu and MSc Duong Manh Cuong that contributed to making this work and had an enjoyable and fulfilling experience

Many thank you and best regards

Student

Luong Tai Chien

CONTENTS

PART I INTRODUCTION 1

1.1 Background and motivation 1

1.1.1 Arthrospira (Spirulina) platensis 1

1.1.1.1 Classification 2

1.1.1.2 Morphology of Arthrospira 3

1.1.1.3 Ultrastructure of A platensis 4

1.1.1.4 The life cycle of Arthrospira (Spirulina) 6

1.1.2 Bioactive peptides of A platensis C1 7

1.1.2.1 Antioxidant and Antiinflammatory activity 7

1.1.2.2 Antihypertension 8

1.1.2.3 Antimicrobial activity 10

1.1.2.4 Antidiabetes and Antiobesity 10

1.1.2.5 Anticancer 10

1.1.3 Computational Tool: Clustal Omega 11

1.2 Objectives 12

1.3 Scope of work 12

PART II MATERIALS AND METHODS 15

2.1 Equipment and Materials 15

2.1.1 Types of equipment 15

2.1.2 Materials 15

2.2 Methods 16

2.2.1 Growing cell A platensis C1 using Zarrouk’s medium [62] 16

2.2.2 Harvesting cell A platensis C1 by membranes filtration 17

2.2.3 Protein extraction from A platensis C1 18

2.2.4 Protein precipitation from A platensis C1 by acetone precipitation 19

2.2.5 Quantitative protein of A platensis C1 by 2-D Quant kit 20

2.2.6 Protein separation from A platensis C1 by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) 21

2.2.7 In silico digestion tools 22

2.2.8 ACE inhibition assay at Institute of Rangsit University 23

2.2.9 Common characteristics of antihypertensive peptides using Clustal Omega 24 PART III RESULTS AND DISCUSSIONS 25

3.1 Culture and cell growth of A platensis C1 25

3.2 Cell harvesting of A platensis C1 26

3.3 Protein extraction of A platensis C1 27

3.4 Protein precipitation from crude extract solution of A platensis C1 27

3.5 Protein quantification of A platensis C1 by 2D-Quant kit 28

3.6 Protein separation from the crude extract of A platensis C1 29

3.7 Common characteristics of antihypertensive peptides from A platensis C1 30

3.8 Discussion 36

PART IV CONCLUSION AND RECOMMENDATIONS 38

4.1 Conclusion 38

4.2 Recommendations 38

LIST OF TABLE

Table 1.1 Amino acid profile of Arthrospira (Spirulina) platensis [60] 1

Table 1.2 Genome statistics of A platensis C1 7

Table 1.3 Examples of bioactive peptides from Arthrospira (Spirulina) [38] 7

Table 2.1 The equipment using for study 15

Table 2.2 The materials using for study 15

Table 2.3 Constituents of Zarrouk’s medium 16

Table 2.4 preparation of the standard curve 20

Table 3.1 The OD480 value of standard protein (BSA)and protein 28

of A platensis C1 28

Table 3.2 Classification of common amino acid of candidate antihypertensive peptides, discovered from A platensis C1 31

Table 3.3 Characteristics of Novel AHP based on ExactAHP_284 and ExactAHP_460 sequences 32

Table 3.4 Amount and percentage of 7 functional amino acid groups of antihypertensive peptides from A platensis C1 34

Table 3.5 Number of amino acids found in Exactly_AHP of A platensis C1 35

Table 3.6 Functional amino acid pattern of Exactly_AHP of A platensis C1 35

LIST OF FIGURES

Fig 1.1 Classification of A platensisusing a cladistic approach [63] 2

Fig 1.2 Morphological aspects of the evident cross-wall in Arthrospira isolated from soda Lake Kailala (Chad) (a) Fresh clonal trichome growth in the laboratory; (b) wild trichome fixed in formaldehyde (2%); (c) fresh clonal trichome grown in mass culture; (d) autofluorescence of trichome shown in (c); Bar maker = 10µm [7] 3

Fig 1.3 Clonal trichomes of Arthrospira indicate isolated from Lake Lonar Arrows indicate the presence of a calyptra Bar marker = 20 mm [7] 4

Fig 1.4 Viewed under a microscope, cells within A platensis trichomes are short and wide and have visible cross-walls [63] 5

Fig 1.5 Life cycle of A platensis [15 6

Fig 1.6 Antihypertension mechanisms of marine algae-derived bioactive peptides that ACE and renin inhibitions are involved [22] 9

Fig 1.7 Screenshot of the Clustal Omega Web page on the EBI Web site: 12

Fig 1.8 Schematic flow work in this report (blue boxes ); yellow boxes, done by Anekthanakul [61] 14

Fig 2.1 Process of cell A platensis culture in Zarrouk’s medium 16

Fig 2.2 Process of harvesting cell A platensis C1 by membrane filtration 17

Fig 2.3 Process of protein extraction from A platensis C1 18

Fig 2.4 Process of protein precipitation from A platensis C1 19

Fig 2.5 Process of quantitative protein of A platensis C1 21

Fig 2.6 Process of the separation protein of A platensis C1 22

Fig 2.7 Process of the in silico digestion and potential 22

Fig 2.8 Process of Antihypertension bioactivity testing 24

Fig 2.9 The Process of discovery for common characteristics of Novel AH peptide of A platensis C1 by Clustal Omega program 24

Fig 3.1 A platensis C1 grown culture in Zarrouk’s medium at35oC, 120 rpm under light illumination of 100 µE/m2/s Grown cells (day, OD560): (A) day 0, 0,1; (B) day 3, 0,742; (C) day 5, 1,463; and (D) day 7, 1,307 25

Fig 3.2 Growth curve of A platensis C1 cultured in Zarrouk’s medium at 35oC,

120 rpm under light illumination of 100 µE/m2/s 26

Fig 3.3 Harvesting of A platensis C1 cells by membranes filtration, (A)

preparation of membranes on the filtration set, (B) adding about 250ml cell of A

platensis C1, (C) harvested cells of A platensis C1 on the membrane before

keeping them at -30oC 27

Fig 3.4 Protein extraction of A platensis C1 cells, (A) after sonicating, (B) after

added dissolve buffer 27

Fig 3.5 Precipitation of crude protein extracted from A platensis C1: (A)

precipitated protein after first centrifugation, (B) after precipitation eight times 28 Fig 3.6 Standard curve of protein standard (BSA) determined by the 2D-Quant kit method 29

Fig 3.7 Protein separation of samples (1 & 2) from A platensis C1 analyzed by

12% SDS-PAGE; (M), Markers 30 Fig 3.8 Common amino acid sequence of candidate antihypertensive peptideswith Novel AHP 33 Fig 3.9 Percentage of 7 functional groups in the amino acid antihypertensive

peptide sequences of A platensis C1 35

LIST OF ABBREVIATION

Electrophoresis

PART I INTRODUCTION 1.1Background and motivation

1.1.1Arthrospira(Spirulina) platensis

Arthrospira (Spirulina)was first described in 1940, by Dangeard[19].However, no report until it was rediscovered by the Belgian botanist,

J Leonard [15].Itis a structured spiral cyanobacteriumbelonging to the group of

blue-greenalgae The well-known species areA platensisand Spirulina maxima

(S.maxima) The algae have the ability to perform photosynthesis like higher

plants, they are found in several environments, including freshwater, seawater and highly alkaline environment particularly in tropical countries[3] The algaecontain very high protein content about 55%-70% (dry weight)which is higher than fish and beef proteins about 3-4 times[49,60], essential fatty acids (18% of total fatty

acid) includesγ-Linolenic acid (GLA), vitamin B12, nucleic acid, minerals such as

chlorophyll.Inside, C-phycocyanin was mostly found as a major part in the algal proteins, which has full of all amino acids(Table 1.1).It alsowas appliedin the food processing functions for human foods and animalfeeds[2]

Table 1.1Amino acid profile of Arthrospira (Spirulina) platensis [60].

This genus is multicellular and a long cylinder

Species: Arthrospira platensis

This species is classified as spirally coiled with granulated cross-walls

Fig 1.1Classification of A platensisusing a cladisticapproach[63].

1.1.1.2 Morphology of Arthrospira

According toSili et al.(2012), the main morphological feature of

Arthrospiraisa typical arrangement of trichome in an open helix usually of relatively

large diameter, sometimes attenuated at the ends, and with evident cross-walls (Fig 1.2) The trichomes are composed of cylindrical cells that undergo binary fission in

a single plane, perpendicular to the main axis Multiplication occurs only by fragmentation of the trichomes The trichome breakage is transcellular and involves the destruction of an intercalary cell[7,42,57]

Fig 1.2 Morphological aspects of the evident cross-wall in Arthrospira isolated from soda Lake Kailala (Chad) (a) Fresh clonal trichome growth in thelaboratory; (b) wild trichome fixed in formaldehyde (2%); (c) fresh clonaltrichome grown in mass culture; (d) autofluorescence of trichome shown in (c); Bar maker = 10µm[7]

The trichome ofA platensishaswidth from about 2,5 to 16 μm, the helix

pitch from about 0 to 80 μm and diameter from 15 to 60 μm These two parameters which define the shape of the helix architecture are highly dependent

on growth and environmental conditions[7].Van Eykelenburg(1980) described

theeffect of temperature on the change trichome structure of A platensisfrom the

helix to the straight in the solid media Although straight and helicoidally forms of

A platensisoften observed in the laboratory However, the factors and the effect

mechanism for this change still remain obscure[58] Jeeji Bai(1985)studied the

effect of the physical and chemical condition on the helix geometry of A

platensis The degree of coiling of trichome from helical to straightform under the

physical and chemical conditions wereregarded as a peculiar property of limb, in the same species and it does not revert back to the helical form [30].The evidence may be due to it is impacted of some mutation in the growth conditions and related

to A platensisagainst photolysis[1,30]

1.1.1.3 Ultrastructure of A platensis

The cell organization of A platensis,observed by electron microscopy shows

organizations such as capsule, pluristratified cell wall, photosynthetic or thylakoid lamella system, ribosomes, and fibrils of DNA region and numerous inclusions is gram-negative [37,48].The capsule has fibrillar structure and covers each filament

protecting it The irregular presence of capsule around the filaments in A platensisis

a differentiating morphological characteristic to compare with S maxima Trichome

width varies from 6 µm to 12 µm and is composed of cylindrical cells, the trichome length is about 500 µm, although in some cases when stirring of culture is deficient the length of filament reaches approximately 1 mm[48] The helix diameter varies from 30 µm to 70 µm [8 ] as shown in Fig 1.3

Fig 1.3Clonal trichomes of Arthrospiraindicate isolated from Lake Lonar Arrows

indicate the presence of a calyptra.Bar marker = 20 mm[7]

Fig 1.4Viewed under a microscope, cells within A platensistrichomes are short

and wide and have visible cross-walls[63]

Arthrospiracell wall is formed by four numbered layers from the

innermost outward as LI, LIT, LIII and LIV layers In 1983,Ciferri reported that all layers are very weak, except layer LII made up of peptidoglycan, a substance that gives to cell wall its rigidity and creates a frame for the cell[15]The LI layer contains β-1,2-glucan, a polysaccharide which is not very digestible for human beings However, the low concentration (<1%) of this layer, the thickness (12 nm), and the protein and lipopolysaccharide nature of the LI layer are favorite

reasons for easier digesting in human digestion [13]

Ciferri(1983),Arthrospira(Spirulina)contains numerous characteristic peripheral

inclusions associated to thylakoids such as polyhedral bodies, cyanophycean granules, polyglucan granules, lipid granules, and polyphosphate granules [11,15]The cyanophycean granules, or reserve granules, are important due to their chemical nature and a series of pigments CO2 has fixated and carry out reserve function by enzyme ribulose 1,5-diphosphate carboxylase at the polyhedral bodies or carboxysomes [48].The polyglucan granules or glycogen granules ora-granules are glucose polymers, small, circular and widely diffused

in the interthylacoidal space The lipid granules, β-granules or osmophilegranules from the reservation deposit, constituted by poly-β-hydroxybutyrate (PHB), found only in prokaryotes [59]

1.1.1.4 The life cycle of Arthrospira (Spirulina)

A fundamental aspect of Arthrospira (Spirulina) biology is its life

cycle (Figure 1.6) due to the taxonomic, physiologic and cultivation implications [15].This period is summarized in three fundamental stages: trichome fragmentation, hormogonia cells enlargement and maturation processes, and trichome elongation The mature trichome are divided into several small filaments or hormogonia through the previous formation of specialized cells, iridium cells, in which the cell material is reabsorbed allowing fragmentation The number of cells in the hormogonia is increased by binary fission and grows lengthwise and takes their helical form [11]

Fig 1.5 Life cycle of A platensis[15]

1.1.1.5 Genome and protein sequences of Arthrospira

Regarding the genome sequencing ofA platensisC1, a strain that has been

studied at KMUTT,the genome contains 6,089,210 bp including 6,108 coding genes and 45 RNA genes, 4,951,337bp DNA coding region, 44,68% of G+C content and no plasmids[16] as summarized in Table 1.2

protein-Table 1.2 Genome statistics of A platensisC1

1.1.2 Bioactive peptides of A platensisC1

Bioactive peptides obtained from A platensisC1have been explored many

biological activities61,such as antioxidant activity [50], antihypertension activity[21], antimicrobial activity, immunomodulatory activity[53], anti-diabetes and anti-obesity activity The peptides are composed of 3 - 20 amino acids and have low molecular weight, about less than 6000 Da [55] with this size, the bioactive peptides will be absorbed quickly into the body through the blood vessels

in mucosa small intestine In general, the bioactive peptides can be synthesizedthrough three basic methods such as enzymatic hydrolysis (as examples shown in Table 1.3), microbial fermentation and solvent extraction [22].Some endogenousbioactive peptides are secondary products from the protein

hydrolysis or even, by external processes mediated by exogenous proteases in

vivo[50]

Table 1.3Examples of bioactive peptides from Arthrospira (Spirulina)[38]

1.1.2.1 Antioxidant and Antiinflammatory activity

Antioxidants are compounds that inhibit the oxidation of other, they have

an important impact on the prevention of different diseases like cardiovascular disease[47].Phycocyanin and β-carotene are two powerful antioxidants and anti-

inflammatory agents found inArthrospira(Spirulina).The first information on the

antioxidant capacity of phycocyanin was introduced in 1998 byRomayet al

(1998) This was also confirmed by subsequent studies[44] Gonzalezet al (1999),Remirez et al.(2002) andRiss et al.(2007)had shown that phycocyanin is

capable of free radical filtration, including alkoxylation, hydroxylation, and peroxyl radicals It also reduces nitrite production, inhibits nitric oxide synthase (iNOS) induction expression, and inhibits lipid peroxidation microsome[26,41,43]

Ge et al.(2006) and Guan et al.(2009)demonstrated the antioxidant activity of

phycocyanin by using phycocyanin recombinant protein technology[25,27]

Some studies have shown the anti-inflammatory effect of phycocyanin inhibits proinflammatory cytokine formation, such as TNFα, suppresses cyclooxygeanase-2 (COX-2) expression and decreases prostaglandin E(2) production [45,40] Besides, phycocyanin has been reported to suppress the activation of nuclear factor-κB (NF-κB) through preventing degradation of cytosolic IκB-α [40]and modulate the mitogen-activated protein kinase (MAPK) activation pathways, including the p38, c-Jun N-terminal kinase (JNK) and extracellular-signal regulated kinase (ERK1/2)[32,36] Studies also showed that β-carotene inhibited the production of nitric oxide and prostaglandin E(2), and suppressed the expression of iNOS, COX-2, TNF-α and IL-1β Such suppression

of inflammatory mediators by β-carotene is likely resulted from its inhibition of NF-κB activation through blocking nuclear translocation of NF-κB p65 subunit

In addition, β-carotene suppressed the transcription of inflammatory cytokines including IL-1β, IL-6, and IL-12 in a macrophage cell line stimulated by lipopolysaccharide (LPS) or IFNγ [31]

1.1.2.2 Antihypertension

Hypertension (HTN or HT), also known as high blood pressure (HBP)is a term referring to statusof the blood pressure in the arteries that is persistently elevated in a long time[4] This is one of the reasons causing heart problems [33] The controlling blood pressure was made by two main systems as kinin-arginine-nitric oxide system and Renin-Angiotensin system (RAS) [17] The angiotensin was created from cutting the inactive peptide angiotensinogen secreted in the liver

by the enzyme renin release from the kidney Subsequently, ACE converts

angiotensin-I into angiotensin-II and inactivates the vasodilator bradykinin, leading

to a rise in blood pressure Thus, there are two ways to control blood pressure by modulating RAS; one is direct inhibition of angiotensin-I generation from angiotensinogen by renin, and another is blockage of conversion from angiotensin-

I to angiotensin-II by ACE (Fig 1.6) Therefore, renin and ACE inhibitory factors are considered as the ways to treat hypertension On the other hand, the kinin-nitric oxide system work in concert with the RAS systems to regulate blood pressure [22]

Fig 1.6Antihypertension mechanisms of marine algae-derived bioactive

peptides that ACE and renin inhibitions are involved[22]

In a study of Suetsuna and Chen, (2001), the peptides derived from A

platensisshowed antihypertensive activity by oral administration in spontaneously

hypertensive rats The peptides fraction (200mg/kg) reduced blood pressure in the

spontaneously hypertensive rats [54.He et al (2007)determined the IC50 values for

ACE inhibitory activity of various hydrolysates including A platensisprotein hydrolysates, and found that ACE inhibitory peptides obtained from A

platensisshowed the lowest IC50 value [29]

1.1.2.3 Antimicrobial activity

Basedon in vitro and in vivostudies,there was reported that phenolic extract from A platensishave ability to inhibit growth of Fusarium graminearum and reproduce mycotoxin, 40% and 62%,respectively [39] Whereas, a study of Sunet

al (2016) which obtained an antibacterial peptide from A platensiswith enzymatic

hydrolysis using alkaline protease and papain enzymes, showed that the minimum

peptide for inhibitory ofEscherichia coli and Staphylococcus aureuswere 8 and 16 mg/ml, respectively [56.Additionally,Saradaet al (2011) showed that gram-positive bacterium Staphylococcus aureus and gram-negative bacteria including E col,

Klebsiella pneumoniae, Pseudomonas aeruginosa were inhibited by C-phycocyanin

from A platensis[49]

1.1.2.4 Antidiabetes and Antiobesity

Report of WHO (2016) showed thatthe world population in 2014, more than 1,9 billion adults were overweight and of these over 600 million were obese.The causes of obesity were related to the digestion enzyme as α-amylase, α-glucosidase, and lipase involved in the digestion of carbohydrates and lipids Briefly, inhibition the above factors play an importantin preventing obesity and

obesity-related diseases.The antidiabetes and antiobesity activities werefound in A

platensisby some studied recently For examples, studies ofVoet al (2015) showed

the water-soluble and water-insoluble of S platensis was effective in lowering the serum glucose level at fasting and glucose loading[9]; In vivostudy evaluating the antidiabetic property of S platensis of Layam and Reddy, (2006),showed diabetic

control by glucose-6-phosphatase activity increased, whereas hexokinase activity

in liver decreased[35]; andin the study ofEl-Desoukiet al (2015)showed an

obvious recovery to theapproximately normal status in diabetic rats treated with

erythropoietin (EPO) and S platensis[20]

1.1.2.5 Anticancer

Chemotherapy is one of the main treatments used to cure cancer, besides that some drugs inhibit the growth of cancer cells have been However, these drugs are often associated with toxicity that causes some side effects such as hair loss, mouth sores, diarrhea, nausea and vomiting, loss of appetite and fatigue [28] The

activity of stimulating antibodies, macrophages, cytokines production, facilitate

lipid and carbohydrate metabolism by S platensis were reported in some studies.Koníckováet al (2014) showed that growth of pancreatic cancer cell were inhibited by S platensis and S platensis-derived tetrapyrroles[34].In the study ofAbu Zaidet al (2015), the IC50(the concentration of A platensisthat is required

to cause 50% inhibition in cell lines viability)for HCT116 and HEPG2 cell lines were 18,8 and 22,3 µg/mLG1,respectively In addition, the correlation between

HCT116, HEPG2 and S platensis water extracts was negative and significant[10]

1.1.3Computational Tool: Clustal Omega

According,Sievers and Higgins, (2014), Clustal Omega (Fig 1.7) are used to create multiple sequences alignments (MSAs).This is a procedure for aligning more than two homologous nucleotide or amino acid sequences together such that the homologous residues from the different sequences lineup as much as possible

in columns This has been one of the most widely used procedures in bioinformatics for decades, as it is an essential prerequisite for most phylogenetic

or comparative analyses of homologous genes or protein[6] Clustal Omega was largely written from scratch using publicly available sequence analysis software libraries such as the SQUID package from Sean Eddy[51It was designed to be able

to align extremely large numbers of sequences very quickly and accurately[6] The speed comes largely from the use of the mBed algorithm [13] The accuracy comes from using Healing, a sophisticated hidden Markov model (HMM) aligner that aligns pairs of HMMs together [52]

Fig 1.7Screenshot of the Clustal Omega Web page on the EBI Web site:

(HTTP:/www.ebi.ac.uk/Tools/msa/clustalo/)

1.2 Objectives

1 To understand and practice forArthrospira (Spirulina) cell cultivation

and harvesting; and for the algal protein extraction and separation

2 To learn and understand computational tool ―SpirPep‖ for the

prediction of putative bioactive peptide sequences of A platensisC1

3 To determine common characteristics ofA platensisC1 antihypertensive

peptides from the computational prediction

1.3 Scope of work

1 Cultivating of A platensisC1 under the optimal cell growth conditions,

and cell harvesting according the protocol of Algal Biotechnology (AGB) lab

@KMUTT

2 The algal protein extraction and quantification using methods as

5.Determining of common characteristics of A platensisC1 antihypertensive

peptides using Clustal Omega

Fig 1.8 Schematic flow work in this report (blue boxes ); yellow boxes, done by

Anekthanakul [61].

PART II MATERIALS AND METHODS

2.1 Equipment and Materials

2.1.1 Types of equipment

Table 2.1 The equipment using for study

Table 2.2 The materials using for study

2.2 Methods

2.2.1 Growing cell A platensisC1 using Zarrouk’smedium[62]

Table 2.3Constituents of Zarrouk’s medium

Fig 2.1 Process of cell A platensis culture in Zarrouk’s medium

- A platensisC1 cultured at 35oC, 120rpm and 100 µE/m2/s(Cell stock)

- The content of cell stock was determined by (OD) spectral measurement

at wavelength 560nm, new Zarrouk’s medium was blank solution

Culture new flasks Shake at 35oC at

Take out new medium same volume

of added cell stock

Add cell stock to new medium

measure OD560of cell stock

Calculate volume cell stock add to new medium

- After analyzed the value of the content of cell stock solution, we continued to calculate the volume of cell stock appropriately to add into the new medium based on the following formula:

C 1 V 1 =C 2 V 2

C 1: is value OD of cell stock

V 1: is the volume of cell stock add to a new medium

C 2 =0.1is initialed OD of new flasks after add cell stock

V 2is the volume of new medium

- After calculated V1, next taking out the volume of the new medium which is similar to the volume of V1

- Added cell stock with volume V1 to new flasks medium

- Culture new flasks cell medium in the incubator and control environmental condition at 35oC, 120 rpm and 100 µE/m2/s

- Observed the growth of cell of A platensis C1 from 1 to 8 days by color

and value of OD560 of cell solution

2.2.2 Harvesting cell A platensis C1 by membranes filtration

Fig 2.2 Process of harvesting cell A platensis C1 by membrane filtration

- The cell of A platensis C1 culture and growth at 35oC, 120rpm and 100 µE/m2/s until the value of OD560 reached 0.4 (mid-log phase) or 4days will

be harvested

- Set up Membranefiltration

Harvesting cells by paper filtration

Wash the cell in paper by 30 ml buffer 5mM HEPES-NaOH pH 7,0 (3 times)

Pick up cell from paper to new tube

Store them at -30oC

- Add 200ml cell solution of A platensis C1 to in the membrane filtration

- Add 30ml buffer to the membrane filtration for cleaning cell A platensis

C1 repeat this step 3 times

- Pick up of A platensis C1 cell from the membrane

- Store them at -30oC

- Note: Prepare buffer 5mM HEPES-NaOH pH 7,0 according to Appendix A

2.2.3 Protein extraction from A platensis C1

Fig 2.3 Process of protein extraction from A platensis C1

- The harvested cell of A platensis C1simultaneously add 30ml buffer 1 in

the tubes (dissolved cell in the buffer by vortex) and transfer to 500ml flask

Dissolve cell in 30 ml Buffer 1

Shake 100 rpm in 1,5 hour

Centrifuge 10,000 rpm at 4oC in 15 min

Discard solution and wash cell with 20ml buffer 2 (2 times)

Dissolve cell with 5 mlbuffer A

Arthrospira platensisC1 cell

Sonicating and checking cell under light microscope

Transfer to 1,5 ml Tube

Centrifuge 10,000 rpm at 4oC in 15 min and remove supernatant

Add 300 µl dissolving solution and mix well

Protein precipitation

- Incubate and shake flask at the 35oC and 120rpm for 1,5hour

- Transfer the cell of A platensis C1from flasks to the tubes 50ml, and

centrifuged at 10,000rpm, 4oC for 15min

- Remove supernatant and add 20ml buffer 2 to tubes and then centrifuged

at 10,000rpm, and 4oC for 15min (repeat this step twice)

- Add buffer A 5ml to tubes

- Transfer sample to tubes 1.5ml and sonicate about 4-6 times (countdown about 10-15 seconds, keep the sample at low temperature by ice during sonicating)

- Checking cell of A platensis C1is completely broken by light

microscope If the cell is not completely, the sonicating step should do again about 1-2 times

and collect a pellet

- Add 300µl dissolving (DS) buffer for dissolving the pellet by vortex until the precipitate completely dissolved

- Note: prepare buffer 1, buffer 2 and bufferA according to Appendix A

2.2.4 Protein precipitation from A platensis C1 by acetone precipitation

Fig 2.4 Process of protein precipitation from A platensis C1

- After dissolved thepellet, next to add 1,2ml Acetone protein precipitation to 300μl protein solution (1 volume of protein solution:4 volume of acetone)

- Store them at temperature -30oC for overnight

Add 1200μl Acetone Keep them in temperature -30oC overnight

Re-precipitate until the pellet is white

300μl protein solution of A platensis C1

- Centrifuge sample If precipitated protein is not white, the pellet will be dissolved with DS buffer and precipitate with acetone and storage at -30oC for overnight until precipitate protein is white

2.2.5 Quantitative protein of A platensis C1 by 2-D Quant kit

- Prepare a standard solution according to Table 2.3 using the 2mg/ml Bovine Serum Albumin (BSA) standard solution provided with the kit

- Prepare tubes containing 1-50µl of the sample to be assayed

- Add 500µl precipitant to each tube (including the standard curve tubes) Vortex briefly and incubate the tubes 2-3min at room temperature

- Add 500µl co-precipitant to each tube and mixing briefly by vortexing or inversion Centrifuge the tube at 10,000g for 5min

- Remove the tubes from the centrifuge as soon as centrifugation is complete A small pellet should be visible Decant the supernatants Proceed rapidly to the next step to avoid resuspension or dispersion of the pellets

- Add 100µl of the copper solution and 400µl water deionized to each tube Vortexing briefly to dissolve the precipitated protein

- Add 1ml of working color (working color reagent by mixing 100 parts color reagent A with 1part color reagent B) to each tube and vortexing each tube Incubate at room temperature for 15-20min

- Reading the absorbance of each tube sample and tubes of the standard at

OD480using water as the reference

- Construction of linear equations

y = ax + b y: values OD480 of the sample

x: protein quantity in the sample

- CalculateCcntent Protein in the sample

Table 2.4preparation of the standard curve

Adding 500 µl precipitant (vortex and incubate at room temperature)

Add 500 µl co-precipitant and mixing, vortex

Centrifuge tube at 10,000g for 5min

Remove supernatant

Add 100 µl copper solution and 400 µl

deionized water to pellet

Add 1ml working color reagent and mix Keep incubate at room temperature

Prepare standard solution

Prepare sample tubes

Read the absorbance of each sample and comparison to the standard curve

Fig 2.5 Process of quantitative protein of A platensis C1

2.2.6 Protein separation from A platensis C1 by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

- The method used in this step according to Gallagher (2007) [23]

- Prepare 12%polyacrylamide gel according to Appendix A

Pouring the separation gel

Pouring the stacking gel

Prepare the sample and load the gel

Running gel at 100v, 20min and 120v, 1h

Coomassie blue staining

Fig 2.6Process of the separation protein of A platensisC1

2.2.7 In silico digestion tools

- Protein sequences of A platensis C1 were subjected to in silico

digestion in order to generate peptide sequences

Fig 2.7 Process of the in silico digestion and potential

antihypertensive peptide prediction [61]

2.2.8 ACE inhibitionassay at Institute of Rangsit University.

The method used in this step according to Chuchote (2015), the Tris buffer (pH 8,3) was used to dilute enzyme and substrate while 10% DMSO in buffer was used to dilute peptide The reaction consisted of 50µl of 2.17 mMHHL, 10µl of 2mU of ACE, and 10µl of 1mg/ml of peptide The HHL and

also incubated at37oC for 10 min before the two solutions were combined and incubated at 37oC withcontinuous agitation at 200 rpm for 30 min The reaction was stopped by adding of 85µl of 1M HCl Prior to HPLC analysis, all solution was filtered through a 0.45µm nylon filter Thefiltered solution was used for the

determination of HA resulting from ACE activity on thesubstrate [18]

10µl 1 mg/ml sample + 50µl 2,17

mM HHL

ACE solution

Incubate 37oC for 10 min

Add ACE to sample

Incubate 37oC for 30 min

Stop reaction by add 1M HCl 85 µl

Filter by syringe filter

Incubate 37oC for 10 min

HPLC analysis

Fig 2.8 Process of Antihypertension bioactivity testing

2.2.9 Common characteristics of antihypertensive peptides using Clustal Omega

The potential antihypertensivepeptide (AH) from Step 2.2.7 and novel antihypertensive peptide (Novel AH) from experiment in Step 2.2.8 in FASTA format were analyzed using Clustal Omega program

- Enter or paste a set of antihypertensive peptide sequences (AH and Novel AH) to the program

- Waiting for the result This tool will return interactive with page of multiple sequences alignment

- Clustering group of antihypertensive peptides by their common amino acid group

- Grouping Novel AH into group of antihypertensive peptides in step 3

- Interpreting the result

Fig 2.9 The Process of discovery for common characteristics of Novel AH

peptide of A platensis C1 by Clustal Omega program

Common characteristics

of both AH and Novel AHpeptide

Submitting peptide sequences to Clustal Omega

Grouping Commonamino acid group The potential antihypertensive peptides (100% identity)in Fasta format

PART III RESULTS AND DISCUSSIONS

3.1 Culture and cell growth of A platensisC1

A platensisC1cells were grownin Zarrouk’s medium at 35oC, 120 rpm under light illumination of 100 µE/m2/s After 3 days, the algal culture changed from light to dark blue-green colorFig 3.1, of which optical density at OD560 nm reached about 0.4-0,5 (mid-log phase) as shown in Fig 3.1 The growing cell until 6 days showed the OD560 reached maximum of 1,463 After 6 days, the culturegrowth

of A platensisC1 was declining (Fig 3.2)

Fig 3.1A platensisC1 grown culture in Zarrouk’s medium at35 o C, 120rpm under light illumination of 100 µE/m 2 /s Grown cells (day, OD 560 ): (A) day 0, 0,1; (B)

day 3,0,742; (C) day 5, 1,463; and (D) day 7, 1,307

Fig 3.2 Growth curve of A platensisC1cultured in Zarrouk’s medium at 35 o C,

120rpm under light illumination of 100 µE/m 2 /s

3.2 Cell harvesting of A platensisC1

The A platensisC1 culture were grown in Zarrouk’s medium at 35oC, 120rpm under light illumination of 100 µE/m2/s until the optical density at OD560

nm reached about 0,4-0,5 (mid-log phase) or after about 2-3 days.After that, the culture was harvested byglass microfiber filters membranes, size about 47 mm The solution of Zarrouk’s medium were removed by suction power of the vacuum machine

Fig 3.3Harvesting of A platensisC1cellsby membranesfiltration, (A) preparation of membranes on the filtration set, (B) adding about 250ml cell of

A platensisC1, (C) harvested cells of A platensisC1on the membrane before

keeping them at -30 o C

3.3 Protein extraction of A platensisC1

The harvested cell of A platensisC1were kept at -30oC until further protein extraction For protein extraction, the algal cells were sonicated on ice The broken cells were passed the extraction process and ready for next step (Fig

3.4)

Fig 3.4Protein extraction of A platensisC1 cells, (A) after sonicating, (B) after

addeddissolve buffer

3.4 Protein precipitation from crude extract solution of A platensisC1

The extracted protein solution was precipitated with acetone The color of the

sample containing protein of A platensisC1 changed from green to

white/non-color after 8 times precipitation The obtained proteinwas quantitativemeasured

by 2D-Quant kit method

Fig 3.5Precipitation of crude protein extracted fromA platensisC1: (A) precipitated proteinafter firstcentrifugation, (B) after precipitationeight times

3.5 Protein quantification of A platensisC1 by 2D-Quant kit

The protein concentrationwas determined by using 2D-Quant Kit The

curve of BSA were plotted as Fig 3.6

Table 3.1 The OD 480 value of standard protein (BSA)and protein

00.10.20.30.40.50.60.70.80.91