Anti-Microbial and Anti-Oxidant Mechanism Study of a Novel Peptide YD1 from Bacillus Amyloliquefaciens
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저작자표시-비영리-변경금지 2.0 대한민국 이용자는 아래의 조건을 따르는 경우에 한하여 자유롭게 l 이 저작물을 복제, 배포, 전송, 전시, 공연 및 방송할 수 있습니다. 다음과 같은 조건을 따라야 합니다: 저작자표시. 귀하는 원저작자를 표시하여야 합니다. 비영리. 귀하는 이 저작물을 영리 목적으로 이용할 수 없습니다. 변경금지. 귀하는 이 저작물을 개작, 변형 또는 가공할 수 없습니다. l 귀하는, 이 저작물의 재이용이나 배포의 경우, 이 저작물에 적용된 이용허락조건 을 명확하게 나타내어야 합니다. l 저작권자로부터 별도의 허가를 받으면 이러한 조건들은 적용되지 않습니다. 저작권법에 따른 이용자의 권리는 위의 내용에 의하여 영향을 받지 않습니다. 이것은 이용허락규약(Legal Code)을 이해하기 쉽게 요약한 것입니다. Disclaimer August 2017 Ph.D. Dissertation Anti-microbial and anti-oxidant mechanism study of a novel peptide YD1 from Bacillus amyloliquefaciens Graduate School of Chosun University College of Pharmacy Md. Saifur Rahman Anti-microbial and anti-oxidant mechanism study of a novel peptide YD1 from Bacillus amyloliquefaciens Bacillus amyloliquefaciens 에서 생산된 펩타이드 YD1 의 항균 및 항산화 작용기작연구 August 25, 2017 Graduate School of Chosun University College of Pharmacy Md. Saifur Rahman Anti-microbial and anti-oxidant mechanism study of a novel peptide YD1 from Bacillus amyloliquefaciens Advisor: Prof. Jin Cheol Yoo This dissertation is submitted to the Graduate School of Chosun University in partial fulfillment of the requirements for the degree of Doctor of Philosophy April 2017 Graduate School of Chosun University College of Pharmacy Md. Saifur Rahman Table of Contents List of Tables vi List of Figures vii Abstract x Abstract (Korean) xiii Abbreviations xvi 1. CHAPTER ONE: INTRODUCTION 01 1.1. Bioactive peptide 02 1.2. Bioactive peptides as pharmaceutical ingredients 04 1.3. Peptide and peptide class 04 1.3.1. Milk peptides 06 1.3.2. Ribosomal peptides 06 1.3.3. Nonribosomal peptides 07 1.3.4. Peptones 07 1.4. Production of peptides 08 1.4.1. Enzymatic hydrolysis 08 1.4.2. Microbial fermentation 11 1.5. Fermented food of Korea 12 1.5.1. Category one 13 1.5.2. Category two 13 1.5.3. Category three 14 1.6. Kimchi fermentation and its microorganisms 15 1.7. Peptide families 21 1.7.1. Opioid peptides 21 i 1.7.2. Immunomodulating peptides 21 1.7.3. Mineral-binding peptides 24 1.7.4. Antithrombotic peptides chain 24 1.7.5. Antimicrobial peptides 24 1.7.6. Antioxidative peptides 26 AIMS OF THE STUDY 27 2. CHAPTER TWO: THE STRAIN CBSYD1 AND PROBIOTIC 28 2.1. INTRODUCTION 29 2.2. MATERIALS AND METHODS 33 2.2.1. Materials 33 2.2.2. Bacterial strain isolation and identification 33 2.2.3. Tolerance towards bile salt 34 2.2.4. Bile Salt plate assay 35 2.2.5. Gastrointestinal tract condition tolerance of Bacillus CBSYD1 35 2.2.6. Bacteriocin activity assay 36 2.2.7. Media optimization 36 2.2.8. Bacteriocin production and protein precipitation 37 2.3. RESULTS AND DISCUSSION 39 2.3.1. Strain isolation and identification 39 2.3.2. Tolerance towards bile salt 39 2.3.3. Bile salt hydrolase 43 2.3.4. Gastrointestinal tract condition tolerance of Bacillus CBSYD1 43 ii 2.3.5. Media optimization 44 2.3.6. Antibacterial activity of Cd.YD1 48 2.4. CONCLUSION 50 3. CHAPTER THREE: ANTIMICROBIAL MECHANISM STUDY 51 3.1. INTRODUCTION 52 3.2. MATERIALS AND METHODS 58 3.2.1. Culture media for YD1 production 58 3.2.2. Antimicrobial activity 58 3.2.3. Purification of YD1 59 3.2.4. Electrophoresis and In-situ analysis 59 3.2.5. Amino acid sequencing and computational analysis 59 3.2.6. Stability of YD1 60 3.2.7. Cytotoxicity 60 3.2.8. Synergism or antagonism of YD1 with antibiotics 60 3.2.9. Time-kill interaction between an antibiotic and YD1 61 3.2.10. Lysis of gram-negative spheroplasts 62 3.2.11. DNA binding assay 62 3.2.12. Transmission electron microscopy (TEM) 63 3.3. RESULTS AND DISCUSSION 64 3.3.1. Media of culture 64 3.3.2. Production, purification, and antimicrobial activity of YD1 65 3.3.3. Effects of YD1 in synergism and E. coli cell 71 3.3.4. Amino acid sequencing, computational analysis, and antimicrobial study 73 iii 3.4. CONCLUSION 83 4. CHAPTER FOUR: ANTIOXIDANT MECHANISM STUDY 84 4.1. INTRODUCTION 85 4.1.1. Environmental stress and factors 86 4.1.2. ROS and its complication in humans 87 4.1.3. Role of exogenous antioxidants in oxidative stress 89 4.1.4. Antioxidant peptides from fermented food kimchi 91 4.2. MATERIALS AND METHODS 93 4.2.1. Radical-scavenging activity assays 93 4.2.1.1. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay 93 4.2.1.2. 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid (ABTS) radical scavenging assay 93 4.2.1.3. Ferric reducing antioxidant power (FRAP) assay 94 4.2.1.4. Cupric-reducing antioxidant capacity (CUPRAC) assay 94 4.2.1.5. Oxygen radical absorbance capacity (ORAC) assay 95 4.2.2. Cell viability 95 4.2.3. Measurement of cellular NO generation 95 4.2.4. Measurement of intracellular ROS generation 96 4.2.5. Preparation of cell lysates and western blot 96 4.2.6. Gene expression and reverse transcription PCR (RT-PCR) analysis 97 iv 4.2.7. Statistics 97 4.3. RESULTS AND DISCUSSION 100 4.3.1. Purified YD1 as an antioxidant 100 4.3.2. Antioxidant activity and mechanism study 101 4.3.2.1. Inhibitory effects of YD1 on cellular NO and ROS generation in RAW 264.7 cells 103 4.3.2.2. Effects of YD1 on gene expression of antioxidants and Phase II antioxidant enzymes 105 4.4. CONCLUSION 115 5. FUTURE DIRECTIONS 116 6. REFERENCES 118 Appendix-1 137 Appendix-2 140 v List of Tables Table 1.1: Examples of non-ribosomal peptides 10 Table 1.2: Microorganisms are used as commercial starters in food fermentation 18 Table 1.3: Some well-known peptides 23 Table 2.1: Anti-bacterial activity of bacteriocin Cd.YD1 49 Table 3.1: Antimicrobial spectrum of YD1 67 Table 3.2: Purification steps of YD1 68 Table 3.3: Average FIC indices resulting from chequerboard titrations between YD1 and selected antibiotics 71 Table 3.4: Alignment between antimicrobial peptides and YD1 75 Table 4.1: Oligonucleotides used in RT-PCR analysis 99 Table 4.2: Comparison of YD1 102 vi List of Figures Figure 1.1: A dipeptide (example Ser-Ala or Ala-Ser) with green marked amino (Alanine) and blue marked carboxyl (Serine). 05 Figure 1.2: A protease regulated hydrolysis of a peptide bond. 09 Figure 1.3: Different fermented food in Korea. 14 Figure 1.4: Fermented food prepared from fish and shellfish and kimchi. 15 Figure 1.5: Microbial communities of 13 kinds of Korean commercial kimchi. 16 Figure 1.6: Structural correlation between met-encefalina. 22 Figure 1.7: Antimicrobial peptides structural classes. 25 Figure 2.1: The schematic diagram demonstrating the potential or known mechanisms of action, whereby probiotic bacteria on the microbiota. 32 Figure 2.2: Phylogenetic tree based on closely complete 16S rRNA gene sequences. 41 Figure 2.3: The culture media without bile salt as a control culture. 42 Figure 2.4: Effects of various nutrition sources in the production of antimicrobial compounds from Bacillus CBSYD1. 46 Figure 2.5: Combined with different percentage nutrients. 47 Figure 3.1: The carpet model, the barrel-stave model and the toroidal model for the antimicrobial peptide-induced killing of bacteria. 53 Figure 3.2: Alternative mechanisms of action of antimicrobial peptides. 54 Figure 3.3: Demonstration of multifunctional activity of antimicrobial peptides. 55 Figure 3.4: Production of bacteriocin and the inhibition activity against Mycobacterium smegmatis ATCC 9341. 66 Figure 3.5: The elution profile of YD1 from a DEAE Sepharose Fast Flow ion exchange column. 69 vii Figure 3.6: Tricine SDS-PAGE electrophoresis and in-situ analysis. 70 Figure 3.7: Effect of YD1 and erythromycin in combination on the rate of kill of E. coli. 72 Figure 3.8: Three-dimensional secondary structure projections of YD1. 73 Figure 3.9: Transmission electron micrographs (TEM) of E. coli cells. 78 Figure 3.10: Percentage of E. coli spheroplasts and total cells remaining intact during exposure to YD1. 79 Figure 3.11: Effects of YD1 on cytotoxicity or cell viability. 80 Figure 3.12: The biological annotations of the target proteins by COACH based on the I-TASSER structure prediction. Predicted ligand binding sites of YD1. 81 Figure 3.13: A diagram of the hypothetical antimicrobial mechanism of action of novel AMP YD1. 82 Figure 4.1: Overview of generation of ROS by various factors. 87 Figure 4.2: Mechanism of antioxidant defenses against free radical-induced damage in humans. 91 Figure 4.3: Radical scavenging effects of YD1 in vitro. 104 Figure 4.4: FRAP assay (B), and CUPRAC assay (C) were conducted with various concentrations of YD1. 106 Figure 4.5: The ORAC activities of the samples were calculated by subtracting the area under the blank curve from the area under the sample curve, to obtain the net area under the curve (Net AUC). 107 Figure 4.6: At a density of 2 × 104 cells per well (96-well plate), RAW 264.7 cells were viii seeded and Cell viability with LPS and YD1 treatment (A) and YD1 treatment alone (B). 107 Figure 4.7: The inhibition of NO and ROS generation in RAW 264.7 cells (A and B). 108 Figure 4.8: Analysis of primary and Phase II antioxidant and detoxifying enzyme mRNA levels in RAW 264.7 cells.