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IN-SILICO EVALUATION OF BIOACTIVITY OF PHYCOCYANIN FROM RED ALGAE
Razia Sultana
Roll No. 0219/15 Registration No. 776
Session: 2019-20
A thesis submitted in the partial fulfillment of the requirements for the degree of Master of Science in Applied Human Nutrition and Dietetics
Department of Applied Food science & Nutrition Faculty of Food Science & Technology
Chattogram Veterinary and Animal Sciences University, Chattogram-4225, Bangladesh
August, 2022
ii | P a g e Authorization
I hereby declare that I am the sole author of the thesis. I also authorize the Chittagong Veterinary and Animal Sciences University (CVASU) to lend this thesis to other institutions or individuals for the purpose of scholarly research. I further authorize the CVASU to reproduce the thesis by photocopying or by other means, in total or in part, at the request of other institutions or individuals for the purpose of scholarly research.
I, the undersigned, and author of this work, declare that the electronic copy of this thesis provided to the CVASU Library, is an accurate copy of the print thesis submitted, within the limits of the technology available
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IN SILICO EVALUATION OF BIOACTIVITY OF PHYCOCYANIN FROM RED ALGAE
Razia Sultana
Roll no: 0219/15 Registration no: 776 Session: 2019-20 (July-Dec)
This is to certify that we have examined the above Master’s thesis and have found that is complete and satisfactory in all respects, and that all revisions required by the thesis examination committee have been made.
………
Dr. Md Abdul Alim (Supervisor)
Department of pathology and parasitology
Faculty of Doctor of Veterinary Medicine, CVASU.
………..
Kazi Nazira Sharmin (Co-investigator)
Department of Applied Food Science and Nutrition
Faculty of Food science and Technology, CVASU
………
Kazi Nazira Sharmin
Chairman of the Examination Committee Department of Applied Food Science and Nutrition
Faculty of Food science and Technology, CVASU
Department of Applied Food science & Nutrition Faculty of Food Science & Technology
Chattogram Veterinary and Animal Sciences University, Chattogram-4225, Bangladesh
August, 2022
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Title of the Thesis: In silico evaluation of bioactivity of phycocyanin from red algae . Name of the student: Razia Sultana
Roll number: 0219/15 Reg. number: 776
Department: Applied Food Science and Nutrition Faculty: Food Science and Technology
Supervisor: Dr. Md Abdul Alim
For office use only
This is to report that as per the check …………. % of the content thesis is stated to be plagiarized and covered/ not covered as per plagiarism policy and institutions issued from CASR, Chattogram Veterinary and Animal Sciences University.
The thesis may/may not be considered for the evaluation.
Dr. Md Abdul Alim Professor
Department of pathology and parasitology Faculty of Doctor of Veterinary Medicine
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DEDICATED to
MY BELOVED PARENTS
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ACKNOWLEDGEMENTS
All credit goes to Almighty Allah for blessing me with the strength, persistence to pursue higher education and complete the thesis for the degree of Masters of Science (MS) in Applied Human Nutrition and Dietetics under the Department of Applied Food Science and technology, at Chattogram Veterinary and Animal Sciences University, Bangladesh.
I pay gratitude to Professor Dr. Goutam Buddha Das, Vice-Chancellor, Chattogram Veterinary and Animal Sciences University (CVASU) for giving special opportunities and providing such research facilities.
I would like to convey my heartfelt gratitude and indebtedness to my supervisor Professor Dr. Md. Abdul Alim, Department of Pathology and Parasitology in Faculty of Veterinary Medicine, at Chattogram Veterinary and Animal Sciences University, Bangladesh, for his advice and suggestions. His unwavering support, insightful recommendations, constructive criticism, guidance encouraged me to persevere throughout this study and thesis preparation.
Also I am very much grateful to Kazi Nazira Sharmin, Head and Associate professor, Department of Applied Food Science and Nutrition, Chattogram Veterinary and Animal Sciences University, Bangladesh, for her befitting enthusiasment, charitable guidance in carrying out the research work.
I would like to express cordial thanks to my loving friends and well-wishers for their cooperation, cheerfulness and inspiration during the course of this study. I also express my heartfelt respects and thanks to my beloved parents and brothers for their understanding, inspirations, support, kindness blessings, and endless love to complete this study.
vii | P a g e TABLE OF CONTENTS
Contents Page No
Authorization……… ii
ACKNOWLEDGEMENTS………... vi
TABLE OF CONTENT………. vii-viii LIST OF TABLES……… ix
LIST OF FIGURES……….. x
LIST OF ABBREVIATION……….. xi-xii ABSTRACT……….. xiii
Chapter 1: INTRODUCTION ………... 1
1.1 Significance of the research………. 3
1.2 Research objectives………. 3
Chapter 2: REVIEW OF LITERATURE………. 4
2.1 Red Algae ………... 4
2.1.1 Characteristics of Red algae……….. 4
2.1.2 Nutritional Composition of Red Algae……….. 5
2.1.3 Health benefits of Bioactive compounds of red algae………... 5
2.2 Red Algae species……… 6
2.3 Red seaweed Protein sequences……….. 6
2.4 Evaluation of Red Algae bioactive peptides through BIOPEP- UWM database……… 7
2.5 In silico proteolysis……… 7
2.6 Virtual screening and characterization of novel tri-peptide……... 7-8 2.7 MALDI-TOF mass spectrometry………... 8-9 2.8 Protein Hydrolysis……….. 9
2.8.1. Factors affecting protein hydrolysis………... 9-10 2.9 Protein Extraction……… 10
2.9.1 Method 1 (urea extraction and acetone precipitation)………... 10-11 2.9.2 Method 2 (urea extraction, ultracentrifugation and acetone precipitation)……… 11
2.9.3 Method 3 (urea extraction, ultracentrifugation, and trichloro acetic acid(TCA) ⁄ acetone precipitation)………. 11
2.9.4 Method 4 (phenol extraction)………... 11 2.9.5 Method 5 (phenol extraction and desalting steps)………. 11-12
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2.10 Bioactive peptides………. 12
2.10.1 Angiotensin-converting enzyme (ACE) inhibitors………….. 12
2.10.2 DPP IV inhibitors……… 12-13 2.10.3 Alpha-glucosidase inhibitors……….. 13
2.11 In silico approach……….. 13-14 2.11.1. Importance of in silico approach……… 14
2.11.2. Advantages and Limitations of in silico approach…………. 14
Chapter 3: MATERIALS AND METHODS……… 15
3.1. Sample for protein sequences………. 15
3.2. Red seaweed protein sequences……….. 15-16 3.3 Evaluation of Red Algae species as potential precursor of bioactive peptides through BIOPEP-UWM database………. 17-19 3.4 In silico proteolysis to release of potential peptides……… 19-21 3.5. Protparam tools………... 21
3.5.1 Peptide Ranker………... 22
3.5.2 Peptide Calculator ………. 22
3.5.3 Bioinformatics tool for allergenicity prediction……… 23
3.5.4 Toxicity Prediction……… 23-24 3.5.5 Peptide Cutter……… 24-25 3.5.6 AHTpin………. 26
Chapter 4: RESULTS ……… 26
4.1 Protein identified by MALDI-TOFF mass Spectrometry………. 26
4.2 Essential amino acid percentage………. 27
4.3 In silico analysis……….. 28-36 Chapter 5: DISCUSSION……….. 37
5.1 Sequences of selected protein……….. 37
5.2 The prospect of red algae as precursor for bioactive peptides……. 37-38 5.3 In Silico proteolysis of red algae protein for phycocyanin………. 38-39 5.4 Virtual Screening and Characterization of Novel peptide………… 39
Chapter 6: CONCLUSION……… 40
Chapter 7: RECOMMENDATIONS & FUTURE PERSPECTIVE.. 41
References..……….. 42-46 Appendix (Profiles for potential biological activity)……… 47 -148 Brief Biography………... 149
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LIST OF TABLES
TABLE TITLE PAGE
2.1 Nutritional composition of Red algae 5 4.1 protein name & accession number used in in silico analysis 26
4.2 Essential amino acid composition 27
4.3 Number of potential bioactive peptides and potential biological activity (B) of identified proteins using BIOPEP
28
4.4 The frequency of occurrence of peptides with a given activity (A) in selected protein sequences
29
4.5.1 The predicted efficiency of release of bioactive fragments from selected red algae protein by in silico proteolysis for ACE inhibitor
30
4.5.2 The predicted efficiency of release of bioactive fragments from selected red algae protein by in silico proteolysis for DPP IV inhibitor
31
4.5.3 The predicted efficiency of release of bioactive fragments from selected red algae protein by in silico proteolysis for Alpha glucosidase inhibitor
32
4.6 Bioactive peptides predicted to be released from red algae protein based on in silico enzymolysis
33
4.7 Predicted potential novel peptide released from red algae
protein 34-36
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LIST OF FIGURES
Figure no. Title Page no.
Figure 3.1 NCBI and FASTA format for protein sequence 15-16 Figure 3.2 Profiles of potential Biological activity 17-18
Figure 3.3 Degree of Hydrolysis 19-20
Figure 3.4 Protparam tool using protein sequence 21 Figure 3.5 PeptideRanker tool for peptide score 22 Figure 3.6 PepCalc for physico-chemical property 22 Figure 3.7 Allertop for Allergenicity prediction 23 Figure 3.8 ToxinPred for toxicity prediction 23-24 Figure 3.9 Determination of resistance of Digestion 24
Figure 3.10 IC50 value Determination 25
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LIST OF ABBREVIATION
ACE Angiotensin-converting enzyme
DPE IV Dipeptidyl peptidase IV
E/S Enzyme to substrate ratio
EU Experimental unit
HCL Hydrochloric acid
KCL Potassium chloride
EDTA Ethylene di-amine tetra acetic acid
Ala Alanine
Arg Arginine
Asn Asparagine
Asp Aspartic acid
Asx Asparagine or aspartic acid
Cys Cysteine
Glu Glutamic acid
Gln Glutamine
Gly Glycine
His Histidine
He Isoleucine
Leu Leucine
Lys Lysine
Met Methionine
Phe Phenylalanine
Pro Proline
Ser Serine
Thr Threonine
Trp Tryptophan
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Tyr Tyrosine
Tyr Tryptophan
Val Valine
kda Kilodalton
% percentage
& And
Et al Et alii/et aliae/et alia
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ABSTRACT
Phycocyanin (PC) is a neutraceutical compound with biological action which is extracted and purified from seaweeds. It is found in red algae, blue-green algae and different seaweeds. According to the Algae Base dynamic species count, there are roughly 10,000 species of seaweeds, the vast majority of which are red algae. The percentage of protein in dried red seaweed varies between 20 and 47 percent (dw), so it’s proteins are potential precursors for showing bioactivity. In this present study, seventeen proteins of red algae (Pyropia endiviifolia, Pyropia pulchra, Neopyropia yezoensis, Neoporphyra haitanensis, Pyropia fucicola, Pyropia kanakaensis, Neoporphyra dentata, Neoporphyra dentata, Galdieria sulphuraria, Polysiphonia urceolata) have been selected as potential precursors of bioactivity based on in silico approach. In silico analysis of Phycocyanin performed high numbers of peptides angiotensin-I-converting enzyme (ACE-I), dipeptidyl peptidase-IV (DPP-IV) and Alpha glucose inhibitor. Chymotrypsin, papain, thermolysin and stem bromelain have been used in-silico proteolysis. For that reason, 45 different tripeptides and dipeptides are tested to see whether any of them can be considered novel bioactive peptides. The distinctive features of the peptides have been explored using Peptide Ranker, PepCalc, Peptide Cutter, ToxinPred, AllerTop, and AHTpin. Bioinformatics analysis indicates that the vast majority of the peptides are likely to be non-toxic, very promising, and safe for use. Future in-vitro and in-vivo studies of the bioactivity of phycocyanin from red algae can be based on these results. This research emphasizes the promise of phycocyanin from red algae as a base material for the creation of novel meals and medicines.
Keywords: Red algae, Phycocyanin, Bioactive peptide, In silico, ACE inhibitor, DPP IV inhibitor, Alpha Glucose inhibitor.
