Biotic Elicitors: Production, Purification, and Characterization

Foreword
Preface
Contents
Contributors
Part I: Protein Compounds as Plant Elicitor
Chapter 1: Production, Purification, and Characterization of Cellulase as Biotic Elicitor
1 Introduction
2 Materials
2.1 Microbial Culture and Media
2.1.1 Cellulase-Producing Strains
2.1.2 Microbiological Media for Culture Maintenance and Cellulase Production (Tables 1, 2, 3, and 4)
2.2 Equipment, Reagents, and Miscellaneous
3 Methods
3.1 Production of Microbial Cellulase Using A. niger
3.1.1 Inoculum Preparation for Fungal Cellulase Production
3.1.2 Production of Cellulase Under Submerged Fermentation Using A. niger
3.1.3 Production of Cellulase Under Solid-State Fermentation Using A. niger
3.1.4 Production of Cellulase Using B. amyloliquefaciens
3.2 Purification of Cellulase Enzyme for Biotic Elicitor Application
3.2.1 Extracellular Cellulase Enzyme Separation from Fermented Broth
Extracellular Cellulase Enzyme Separation from Solid-State Fermentation Samples
Extracellular Cellulase Enzyme Separation from Submerged Fermentation Samples
3.2.2 Extraction and Purification from Cell-/Mycelium-Free Supernatant [11]
3.2.3 Characterization of Cellulase
Relative Molecular Weight Determination of Cellulase
Zymography of Cellulase
Temperature and pH Stability of Cellulase
4 Notes
References
Chapter 2: Production, Purification, and Characterization of Elicitin from Phytophthora Species
1 Introduction
2 Materials
2.1 Fungal Culture and Maintenance (see Note 1)
2.2 Production and Partial Purification of Culture Filtrate
2.3 Elicitin Purification Using Chromatographic Procedure
2.4 Characterization of Elicitin
3 Methods
3.1 Fungal Cultures and Growth Conditions
3.2 Production and Partial Purification of the Culture Filtrate
3.3 Elicitin Purification Using a Chromatographic Procedure
3.4 Characterization of Elicitin on Tris/Tricine Gel
4 Note
References
Chapter 3: Production, Purification, and Characterization of Oligandrin from Pythium oligandrum
1 Introduction
2 Materials
2.1 Fungal Culture
2.2 Production of the Culture Filtrates of Pythium oligandrum
2.3 Oligandrin Purification and Characterization from the Culture Filtrates
2.4 SDS Polyacrylamide Gel
3 Methods
3.1 Fungal Cultures and Growth Conditions
3.2 Production of the Culture Filtrates of Pythium oligandrum
3.3 Oligandrin Purification and Characterization from the Culture Filtrates
3.4 SDS Polyacrylamide Gel
4 Notes
References
Chapter 4: Production, Purification, and Characterization of Endoxylanase
1 Introduction
2 Materials
2.1 Xylanase Production on Solid-State Fermentation (SSF)
2.2 Purification of Xylanase
2.3 Enzymatic Activity
3 Methods
3.1 Production of Xylanase (SSF)
3.2 Purification of Xylanase
3.3 Characterization of Xylanase
3.3.1 Measurement of Xylanase Activity
3.3.2 Molecular Weight Determination
3.4 Effect of pH on Activity and Stability
3.5 Screening of Xylanolytic Strains
4 Notes
References
Chapter 5: Flagellin
1 Introduction
2 Materials
2.1 Extraction of Flagellin
2.2 Purification of Flagellin by Anion Exchange Chromatography, Ultrafiltration, and SDS Gel Electrophoresis
2.3 Characterization of Flagellin Using MALDI TOF/TOF
3 Methods
3.1 Extraction of Flagellin
3.2 Purification of Flagellin by Anion Exchange Chromatography, Ultrafiltration, and SDS Gel Electrophoresis
3.3 Characterization of Flagellin Using MALDI TOF/TOF
4 Notes
References
Chapter 6: Victorin
1 Introduction
2 Materials
2.1 Production of Crude Victorin Toxin from Helminthosporium victoriae Culture
2.2 Detection of Victorin in Crude Culture Fluids with a SEP-PAK Cartridge
2.3 Partial Purification of Victorin Using Column Chromatography
2.4 Partial Purification of Victorin from Culture Fluids of Isolate Hv-1 by Thin Layer Chromatography (TLC)
2.5 Purification of Victorin Using HPLC
3 Methods
3.1 Production of Crude Victorin Toxin from Helminthosporium victoriae Culture
3.2 Detection of Victorin in Crude Culture Fluids with a SEP-PAK Cartridge
3.3 Partial Purification of Victorin Using Column Chromatography
3.4 Partial Purification of Victorin from Culture Fluids of Isolate Hv-1 by Thin Layer Chromatography
3.5 Purification of Victorin Using HPLC
4 Notes
References
Chapter 7: Chitinase
1 Introduction
2 Materials
2.1 Culture Preparation
2.2 Chitinase Enzyme Activity
2.3 Screening for Chitin Degradation
2.4 Purification of Chitinase
2.5 Determination of Protein Concentration
2.6 Sodium Dodecyl Sulfate Polyacrylamide (SDS-PAGE) Gel Electrophoresis
2.7 Characteristics of Crude and Purified Chitinase
3 Methods
3.1 Culture Preparation
3.2 Chitinase Enzyme Activity
3.3 Screening for Chitin Degradation
3.4 Purification of Chitinase (see Note 3)
3.4.1 Ammonium Sulfate Precipitation
3.4.2 Chitin Affinity Chromatography
3.5 Determination of Protein Concentration
3.6 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
3.6.1 Zymogram Analysis of Chitinase
3.7 Characteristics of Crude and Purify Chitinase
3.7.1 Temperature and pH Stability of the Enzyme
3.7.2 Antifungal Activity of Crude and Purified Chitinase Sample
4 Notes
References
Part II: Carbohydrate Compounds as Plant Elicitor
Chapter 8: Production, Purification, and Characterization of Carbohydrate Elicitor: β-Linked Glucopyranosyl
1 Introduction
2 Materials
2.1 Production of β-D-Glucopyranosyl-D-Glucitol
2.1.1 Pmg Culture Condition
2.1.2 Preparation of Pmg Mycelial Walls (see item 1)
2.1.3 Preparation of Pmg Mycelial Walls (see item 2)
2.1.4 Partial Acid Hydrolysis
2.2 Purification of β-D-Glucopyranosyl-D-Glucitol
2.2.1 Extraction of Elicitor from Purified Mycelial Walls of Pmg
2.2.2 Fractionation of Wall-Released Elicitor
2.2.3 Purification
Low-Resolution Gel Filtration Chromatography
High-Resolution Gel Filtration Chromatography
NaBH4 Reduction of Oligoglucosides
High-Performance Liquid Chromatography
Normal-Phase Partition Chromatography
Reversed-Phase Liquid Chromatography
2.3 Characterization of β-D-Glucopyranosyl-D-Glucitol
2.3.1 Determination of the Absolute Configuration of the Glycosyl Residues of Oligoglucosyl-Glucitol Fractions
2.3.2 Glycosyl Linkage Composition of the Oligoglucosyl-Glucitols
2.3.3 H-NMR
2.3.4 FAB-MS
3 Methods
3.1 Production of β-D-Glucopyranosyl-D-Glucitol
3.1.1 Pmg Culture Condition
3.1.2 Preparation of Pmg Mycelial Walls (see item 1)
3.1.3 Preparation of Pmg Mycelial Walls (see item 2)
3.1.4 Partial Acid Hydrolysis
3.2 Purification of β-D-Glucopyranosyl-D-Glucitol
3.2.1 Extraction of an Elicitor from Purified Mycelial Walls of Pmg
3.2.2 Fractionation of Wall-Released Elicitor
3.2.3 Purification
Low-Resolution Gel Filtration Chromatography
High-Resolution Gel Filtration Chromatography
NaBH4 Reduction of Oligoglucosides
High-Performance Liquid Chromatography
Normal-Phase Partition Chromatography
Reversed-Phase Liquid Chromatography
3.3 Characterization of β-D-Glucopyranosyl-D-Glucitol
3.3.1 Determination of the Absolute Configuration of the Glycosyl Residues of Oligoglucosyl-Glucitol Fractions
3.3.2 Glycosyl Linkage Composition of the Oligoglucosyl-Glucitols
3.3.3 H-NMR
3.3.4 FAB-MS
4 Note
References
Chapter 9: Chitosan
1 Introduction
2 Materials
2.1 Extraction of Chitin from Crustaceans and Production of Chitosan
2.2 Characterization of Chitosan
2.2.1 Deacetylation of Chitin
2.3 Measurement of Degree of Deacetylation
2.3.1 Acid-Base Titration [10]
2.3.2 Potentiometric Titration
2.4 Microscopic Method
2.4.1 Scanning Electron Microscopy
2.5 Chromatographic Methods for Detection of Chitosan
2.5.1 HPLC Analysis: Acid Hydrolysis
2.5.2 GC (Gas Chromatography) Analysis: Pyrolysis
2.6 Spectroscopic Method for Detection of Chitosan
2.6.1 FTIR (Fourier Transform Infrared Spectroscopy) Analysis
3 Methods
3.1 Extraction of Chitin from Crustaceans and Production of Chitosan
3.2 Extraction of Chitin from Fungi
3.3 Characterization of Chitosan
3.3.1 Deacetylation of Chitin
3.4 Measurement of Degree of Deacetylation
3.4.1 Acid-Base Titration
3.4.2 Potentiometric Titration
3.5 Microscopic Method
3.5.1 Scanning Electron Microscopy
3.6 Chromatographic Methods for Detection of Chitosan
3.6.1 HPLC Analysis: Acid Hydrolysis
3.6.2 GC (Gas Chromatography) Analysis: Pyrolysis
3.7 Spectroscopic Method for Detection of Chitosan
3.7.1 FTIR (Fourier Transform Infrared Spectroscopy) Analysis [14]
4 Notes
References
Chapter 10: Production, Purification, and Characterization of Carbohydrate Elicitor: Pectic Oligomers
1 Introduction
2 Materials
2.1 Sample Preparation
2.2 Alcohol-Insoluble Solid (AIS) Preparation
2.3 Polysaccharide Isolation
2.4 Assessment of Enzymatic Activities of Selected Multi-Enzymatic Preparations
2.5 Enzymatic Fragmentation of Pectic Polysaccharides
2.6 Characterization of Structural Properties
2.6.1 Sugar Content and Monosaccharide Profiling
2.6.2 Phenol-Sulfuric Acid Method for Total Carbohydrates
2.6.3 Molecular Weight Distribution Analysis
2.7 Preparation of Methylated PMP-Monosaccharides
2.8 Sugar Linkage Analysis by Liquid Chromatography-Mass Spectrometry
3 Methods
3.1 Sample Preparation
3.2 Alcohol-Insoluble Solid (AIS) Preparation
3.3 Polysaccharide Isolation
3.4 Assessment of Enzymatic Activities of Selected Multi-Enzymatic Preparations
3.5 Enzymatic Fragmentation of Pectic Polysaccharides
3.6 Characterization of Structural Properties
3.6.1 Sugar Content and Monosaccharide Profiling
3.6.2 Molecular Weight Distribution Analysis
3.7 Preparation of Methylated PMP-Monosaccharides
3.8 Sugar Linkage Analysis by Liquid Chromatography-Mass Spectrometry
4 Notes
References
Chapter 11: Chitin: Production, Purification, and Characterization
1 Introduction
2 Materials
2.1 Raw Materials
3 Methods
3.1 Chemical Process
3.1.1 Protocol 1: Chitin Preparation from Shells of Shrimp
Homogenized Powder Preparation
Demineralization
Deproteinization
Deacetylation (Optional)
3.1.2 Protocol 2: Chitin Extraction from Shrimp Shell Waste
Homogenized Powder Preparation
Demineralization
Deproteinization
Deacetylation (Optional)
3.1.3 Protocol 3: Chitin Extraction from Crustacean Waste
Homogenized Powder Preparation
Demineralization
Deproteinization
Deacetylation (Optional)
3.1.4 Protocols 1-4: Depigmentation of Chitin
3.2 Biological Process
3.2.1 Protocol 4: Chitin Extraction from Bacterial Strains
Preparation of Bacterial Strains and Inoculums
Fermentation Conditions for DM and DP
Deacetylation (Optional)
3.3 Characterization
3.3.1 Sulfated Ash Content
3.3.2 Infrared Spectra (IR) Analysis
Protocol 1: Fourier Transform Infrared Spectroscopy (FTIR)
3.3.3 Elemental Analysis
Protocol 1: Elemental Analysis Using Elemental Analyzer
3.3.4 X-Ray Diffraction
4 Notes
References
Chapter 12: Laminarin
1 Introduction
2 Materials
2.1 Source
2.2 Extraction
3 Methods
3.1 Sample Collection
3.2 Extraction
3.2.1 Method 1: Ultrasound-Assisted Extraction
3.2.2 Method 2: Hydrothermal-Assisted Extraction (HAE)
3.3 Purification
3.4 Characterization
3.4.1 Quantification
Method 1: Identification and Quantification by HPLC
Method 2: By UV Spectrophotometer
3.4.2 Fourier Transform Infrared Spectroscopy (FTIR)
3.4.3 Quadrupole Time-of-Flight Mass Spectrometry (Q-TOF-MS)
References
Chapter 13: Production, Extraction, and Characterization of Mannan/Mannan Oligosaccharides (MOS)
1 Introduction
2 Materials
2.1 Microbial Culture and Production Media
2.2 Equipment, Reagents, and Miscellaneous Requirement
3 Methods
3.1 Growth of Saccharomyces cerevisiae for Mannan/MOS Production
3.2 Extraction of Crude Mannan (MOS)
3.3 Deproteinization of Mannan Oligosaccharides (MOS)
3.3.1 The Sevage Method
3.3.2 The TCA Method
3.3.3 The Hydrochloric Acid Method
3.4 Quantitative Estimation of Mannan Oligosaccharides (MOS)
3.5 Characterization of Mannan Oligosaccharides (MOS)
3.5.1 FT-IR Analysis
3.5.2 Thin Layer Chromatography (TLC) Analysis
3.6 Assessment of the Elicitor Activity of Mannan/MOS
4 Notes
References
Chapter 14: Microbial β-Glucan: Production, Extraction, and Characterization
1 Introduction
2 Materials
2.1 Microbial Culture and Production Media
2.2 Equipment, Reagents, and Miscellaneous Requirement
3 Methods
3.1 Isolation of Glucan-Producing Microorganism
3.2 Glucan Production
3.2.1 Preparation of Inoculum
3.2.2 Production Media (Mineral Salt Medium) (Tables 1, 2, and 3)
3.3 Extraction/Purification of Glucan
3.4 Characterization of Glucan/EPS
3.4.1 Thin Layer Chromatography (TLC) Analysis
3.4.2 Fourier Transform Infrared (FT-IR) Spectrometry Analysis
3.4.3 NMR and Particle Size Analysis
3.4.4 Aniline Blue Staining
3.5 Assessment of the Elicitor Activity of Glucan
4 Notes
References
Part III: Lipid Compounds as Plant Elicitor
Chapter 15: Fatty Acid Amino Acid Conjugates (FACs)
1 Introduction
2 Materials
2.1 Gut Content Extraction
2.1.1 Caterpillar Source and Rearing
2.1.2 Extraction Procedure
2.2 Identification of FACs
2.2.1 LC-MS Analyses
2.2.2 In Vitro Biosynthesis of FACs with Enzyme Extracts
2.2.3 Synthesis of 15N-Labeled FACs and Incubation Experiments
2.2.4 Amino Acid Analyses
3 Methods
3.1 Gut Content Extraction
3.1.1 Caterpillar Source and Rearing
3.1.2 Extraction Procedure
3.2 Identification of FACs
3.2.1 LC-MS Analyses
3.2.2 In Vitro Biosynthesis of FACs with Enzyme Extracts
Sample Preparation
Biosynthesis of FACs
3.2.3 Synthesis of 15N-Labeled FACs and Incubation Experiments
3.2.4 Amino Acid Analyses
4 Notes
References
Chapter 16: Ergosterol
1 Introduction
2 Materials
2.1 Analysis of Ergosterol in Agaricus bisporus
2.2 Extraction and Purification of Ergosterol in Agaricus bisporus
2.3 HPLC Analysis
3 Methods
3.1 Analysis of Ergosterol in Agaricus bisporus
3.1.1 Sterol Extraction and Saponification
3.2 Extraction and Purification of Ergosterol in Agaricus bisporus
3.3 HPLC Analysis
4 Notes
References
Chapter 17: Coronatine
1 Introduction
2 Materials
2.1 Culture of Pseudomonas syringae
2.2 Extraction of Coronatine
2.3 HPLC Analysis
3 Methods
3.1 Culture of Pseudomonas syringae
3.2 Extraction of Coronatine
3.3 HPLC Analysis
4 Notes
References
Chapter 18: Fumonisin B1
1 Introduction
2 Materials
2.1 Production of Crude FB1 from Fusarium proliferatum Culture
2.2 Purification of Isolated FB1 by XAD-16 Adsorption LC
2.3 Purification of FB1 by DEAE-Sepharose Ion Exchange Chromatography
2.4 Purification of FB1 by C18 Partition LC
3 Methods
3.1 Production of Crude FB1 from Fusarium proliferatum Culture
3.2 Purification of FB1 by Chromatographic Techniques
3.2.1 Purification of FB1 by XAD-16 Adsorption LC
3.2.2 Purification of FB1 by DEAE-Sepharose Ion Exchange LC
3.2.3 Purification of FB1 by C18 Partition LC
3.3 Characterization of FB1
3.3.1 Characterize FB1 by TLC
3.3.2 HPLC
3.4 Storage
4 Note
References
Part IV: Other Compounds as Plant Elicitor
Chapter 19: Glycoprotein
1 Introduction
2 Materials
2.1 Culture Preparation
2.2 Purification
2.2.1 SDS-PAGE Analysis
2.3 Identification of Glycoprotein
2.3.1 Content of Protein and Carbohydrate in Glycoprotein
2.3.2 Deglycosylation Analysis with Chemical Method of Trifluoromethane-Sulfonic Acid
2.3.3 High-Performance Liquid Chromatography (HPLC)
2.3.4 Leaf Bioassay
3 Methods
3.1 Culture Preparation
3.2 Purification (See Note 2)
3.3 Identification of Glycoprotein
3.3.1 SDS-PAGE Analysis (See Note 3)
For Protein Detection
For Glycoprotein Detection (See Note 4)
3.3.2 Content of Protein and Carbohydrate in Glycoprotein
3.3.3 Deglycosylation Analysis with Chemical Method of Trifluoromethane-Sulfonic Acid
3.3.4 High-Performance Liquid Chromatography (HPLC)
3.3.5 Leaf Bioassay
4 Notes
References
Chapter 20: Polygalacturonase
1 Introduction
2 Materials
2.1 Fungal Culture and Maintenance
2.2 Solid-State Fermentation for Polygalacturonase Production
2.3 Enzyme Assay and Protein Estimation
2.4 Enzyme Purification
2.5 Native PAGE and Zymogram Study
2.6 SDS-PAGE
3 Methods
3.1 Fungal Culture and Maintenance
3.2 Solid-State Fermentation for Polygalacturonase Production
3.3 Enzyme Assay and Protein Estimation
3.3.1 Sample Preparation
3.3.2 Enzyme Assay
3.4 Enzyme Purification
3.5 Native PAGE and Zymogram Study
3.6 SDS-PAGE
4 Notes
References
Chapter 21: Production, Purification, and Characterization of Glycolipid as Biotic Elicitor
1 Introduction
2 Materials
2.1 Microbial Culture and Media
2.1.1 Glycolipid-Producing Strains
2.1.2 Microbiological Media and Culture Maintenance for Glycolipid Production
2.2 Equipment, Reagents, and Miscellaneous
3 Methods
3.1 Glycolipid-Producing Strain Maintenance and Inoculum Development
3.1.1 Culture Maintenance and Seed Culture Development of C. lipolytica
3.1.2 Culture Maintenance and Seed Culture Development of Fungal Strain U. maydis FBD12
3.1.3 Culture Maintenance and Inoculum Preparation of Pseudomonas sp. LM19
3.2 Microbial Production of Glycolipids
3.2.1 Glycolipid Production Using C. lipolytica
3.2.2 Glycolipid Production Using Fungal Strain U. maydis FBD12
3.2.3 Glycolipid Production Using Pseudomonas sp. LM19
3.3 Extraction and Purification of Glycolipids for Biotic Elicitation
3.3.1 Cell-Free Supernatant
3.3.2 Precipitation of Glycolipids
Acid Precipitation of Glycolipid
Solvent Extraction of Glycolipid
Large-Scale Glycolipid Extraction by Ethyl Acetate
3.3.3 Purification of Glycolipids by Column Chromatography
3.4 Characterization of Microbial Glycolipids
3.4.1 Characterization of Glycolipid by TLC
3.4.2 Characterization of Glycolipid by Fourier Transform Infrared Spectroscopy (FTIR)
3.4.3 Estimation of Glycolipids by HPLC
3.4.4 Quantification of Glycolipid by GC-MS
3.4.5 Characterization of Glycolipid by NMR
4 Notes
References
Index