Multienzymatic Assemblies: Methods and Protocols

Description

This volume details cutting-edge methods and protocols for the development, characterization, and applications of multienzyme assemblies. Chapters guide readers through up-to-date techniques applied for the development and emerging applications of multi-enzymatic systems for biotransformations, biosensing, molecular-scale diagnostics and bioelectronics. Written in the format of the highly successful Methods in Molecular Biology series, each chapter includes an introduction to the topic, lists necessary materials and reagents, includes tips on troubleshooting and known pitfalls, and step-by-step, readily reproducible protocols. Authoritative and cutting-edge, Multienzymatic Assemblies: Methods and Protocols aims to be useful for academics and industry professionals working in the field of biotechnology, biochemistry, chemical and biological engineering, nanobiotechnology, and biocatalysis. Preface Contents Contributors Chapter 1: Recombinant Expression and Purification of Large Bacterial Multienzyme Assemblies for Biosynthetic Processes 1 Introduction 2 Materials 2.1 Expression 2.2 Cell Lysis and His-tag Purification 2.3 FLAG-tag Purification 2.4 Gel Filtration 3 Methods 3.1 Protein Expression 3.2 His-tag Affinity Column 3.3 FLAG-tag Affinity Column (See Note 21) 3.4 Gel Filtration Column 4 Notes References Chapter 2: Multi-dimensional Fluorescence Live-Cell Imaging for Glucosome Dynamics in Living Human Cells 1 Introduction 2 Materials 2.1 Human Cancer Cell Lines 2.2 Cell Culture and Imaging 3 Methods 3.1 Human Cell Culture 3.2 Transient Transfection 3.3 2-Dimentional (2D) Visualization of Glucosome Using Wide-Field or Confocal Microscopy 3.4 3D Visualization of Glucosome Using Lattice Light-Sheet Microscopy (LLSM) 4 Notes References Chapter 3: Mechanisms and Effects of Substrate Channelling in Enzymatic Cascades 1 Introduction 2 Types and Mechanisms of Channelling 2.1 Multifunctional Enzymes 2.1.1 Molecular Channels 2.1.2 Electrostatic Channelling 2.2 Metabolons and Enzyme Clusters 2.3 Synthetic Enzyme Complexes 3 Effects of Channelling 3.1 Acceleration/Deceleration of Reaction Cascades 3.1.1 Direct Channelling 3.1.2 Enzyme Proximity 3.2 Protection of Intermediates 3.2.1 Direct Channelling 3.2.2 Enzyme Proximity 3.3 Protection of Surroundings 4 Macromolecular Crowding 5 Concluding Remarks References Chapter 4: Structural Characterization of Multienzyme Assemblies: An Overview 1 Introduction 2 From Single Enzymes to Complex Multienzyme Structures 3 Mechanistic Details of Multienzyme Assemblies 3.1 Electrostatic Guidance 3.2 Tunneling 3.3 Swinging Arms 4 Artificial Multienzyme Assemblies 5 Emerging X-ray Crystallography Techniques 6 Conclusions References Chapter 5: Assessing Protein Interactions for Clustering of Mitochondrial Urea Cycle Enzymes 1 Introduction 2 Materials 2.1 Isolation of Mitochondria by Differential Fractionation (See Note 1) 2.2 Protein Determination 2.3 Co-immunoprecipitation 2.4 Fractionation of Rat Liver Mitochondria 2.5 Immunoblotting 2.6 Culturing Mouse Primary Hepatocytes on Coverslips 2.7 Immunofluorescence 2.8 Confocal and Super Resolution (gSTED) Microscopy 3 Methods 3.1 Co-immunoprecipitation of Mitochondrial Urea Cycle Enzymes from Liver Mitochondria 3.2 Biochemical Co-localization of Mitochondrial Urea Cycle Enzymes at the IMM 3.3 Super Resolution Imaging of Mitochondrial Urea Cycle Enzymes at the IMM 3.4 Confocal Microscopy 3.5 Confocal-gSTED Microscopy 3.6 Image Processing 4 Notes References Chapter 6: DNA Nanoscaffolds for Multienzyme Systems Assembly 1 Introduction 2 Materials 2.1 Common Stock Buffer 2.2 Gel Electrophoresis Supplies 2.3 Denature Polyacrylamide Gel Electrophoresis (PAGE) 2.4 DNA-Cofactor Bioconjugation 2.5 DNA-Enzyme Bioconjugation 2.6 Non-denature PAGE 2.7 AFM Characterization 2.8 Enzyme Activity Characterization 3 Methods 3.1 Design of DNA Nanoscaffolds 3.2 Denature PAGE for Oligonucleotide purification 3.2.1 Gel Casting Assembly of Denature PAGE Gel 3.2.2 Preparation of Denature PAGE 3.2.3 Preparation of Samples for Oligonucleotide Purification 3.2.4 Collect Oligonucleotide from Denature PAGE 3.3 DNA-Cofactor Bioconjugation 3.3.1 DNA-Cofactor Bioconjugation 3.3.2 HPLC Purification of Cofactor-Conjugated DNA 3.4 DNA-Enzyme Conjugation 3.4.1 Conjugation of PROTEIN-SPDP with thiol-DNA (Fig. 3) 3.4.2 DNA-protein Conjugation 3.4.3 Anion-exchange Purification of DNA-conjugated Protein 3.5 Assembly of Enzymes on DNA Nanoscaffolds 3.5.1 DNA Nanoscaffold Assembly 3.5.2 Enzyme Assembly on DNA Nanoscaffold 3.6 Non-denature PAGE to Characterize Small DNA Assemblies 3.7 AFM Characterization 3.7.1 Mica Preparation 3.7.2 Image Nanostructures by SCAN AYST in Fluid 3.7.3 AFM Image Analysis 3.8 Enzyme Activity Characterization 4 Notes References Chapter 7: Strategies for Multienzyme Assemblies 1 Introduction 1.1 Cellulosome-Based Enzyme Assemblies 1.2 Protein Nanoparticle Immobilization Strategies 1.2.1 Sortase-Mediated Ligation 1.2.2 Spytag/SpyCatcher Bioconjugation 1.3 Protein-Nucleic Acid Interaction Strategies 1.3.1 HaloTag Mediated Protein-DNA Labeling 1.3.2 Zinc Finger Protein Enabled Protein Immobilization on DNA Template 1.3.3 Strand Displacement for Enzyme Function Programming 2 Materials 2.1 Cellulosome-Based Enzyme Assemblies 2.1.1 Culture Medium 2.1.2 Protein Expression and Cell Lysis 2.1.3 Immunofluorescence Microscope 2.2 Sortase-Mediated and Spytag/SpyCatcher Bioconjugation 2.2.1 Protein Expression and Purification 2.2.2 Sortase Reaction 2.2.3 Spytag/SpyCatcher Reaction 2.2.4 Inverse Transition Cycling (ITC) 2.2.5 SDS-PAGE 2.2.6 Equipment 2.3 HaloTag Mediated Protein-DNA Labeling 2.3.1 Equipment 2.3.2 Buffers and Reagents 2.4 Zinc Finger Protein Enabled Protein Immobilization on DNA Template 2.4.1 Equipment 2.4.2 Buffers and Reagents 2.5 Strand Displacement for Enzyme Function Programming 2.5.1 Equipment 2.5.2 Buffers and Reagents 3 Methods 3.1 Cellulosome-Based Enzyme Assemblies 3.1.1 Cellulase Expression and Cell Lysis 3.1.2 Display of Scaffoldin on Yeast Surface 3.1.3 Mini-cellulosome Assembly and Immunofluorescence Microscopy 3.2 Sortase-Mediated and Spytag/SpyCatcher Conjugation 3.2.1 Transformation of pET Plasmid into BL21 E. coli Cells 3.2.2 Sortase and Target Protein Expression (IPTG Induction) 3.2.3 ELP-LPETG Protein Expression (Leaky Expression) 3.2.4 Sortase Reaction 3.2.5 Spytag/SpyCatcher Bioconjugation 3.2.6 ELP Purification 3.2.7 SDS-PAGE 3.3 HaloTag Mediated Protein-DNA Labeling 3.4 Zinc Finger Protein Enabled Protein Immobilization on DNA Template 3.5 Strand Displacement for Enzyme Function Programming 4 Notes References Chapter 8: Random and Positional Immobilization of Multi-enzyme Systems 1 Introduction 1.1 Random Co-immobilization 1.2 Positional Co-immobilization 2 Materials 2.1 Random Co-immobilization 2.2 Positional Co-immobilization 3 Methods 3.1 Random Co-immobilization 3.1.1 Preparation of GO 3.1.2 Preparation of rGO 3.1.3 Enzyme Immobilization 3.2 Positional Co-immobilization 3.2.1 Methods Used on Inorganic Structure Biotinylation De-PG2-mediated GOx Immobilization onto Sensor Chips De-PG2-mediated Enzyme Immobilization Inside Glass Tubes HRP and GOx Activity Measurements Activity of Dissolved GOx Activity of Immobilized GOx D-Glucose Quantification Via the GOx/HRP Cascade Reaction 3.2.2 Methods Used on DNA Structure Lipase Activity Measurements Enzyme Immobilization, Removal, and Re-hybridization Activity Determination of Immobilized DNA-CalB Three-Enzyme Cascade Reaction 4 Notes References Chapter 9: Compartmentalized Immobilization of Multi-enzyme Systems 1 Introduction 2 Materials 3 Methods 3.1 Compartmentalized Multi-enzyme Immobilization on Inorganic Complexes 3.2 Protein Scaffold for Enzyme Compartmentalization 3.3 Nucleic Acid Scaffold for Enzyme Compartmentalization 3.3.1 Pathway Plasmid Construction 4 Notes References Chapter 10: 3D Printed Polylactic Acid Well-Plate for Multi-enzyme Immobilization 1 Introduction 2 Materials 2.1 Equipment 2.2 Three-Dimensional (3D) Design and Printing of Polylactic Acid (PLA) Well-Plates 2.3 Surface Modification and Enzyme Immobilization on 3D Printed PLA Well-Plates 2.4 Enzymatic Activity Measurement in the PLA Well-Plates 3 Methods 3.1 3D Design of a PLA Well-Plate 3.1.1 2D Sketch Design 3.1.2 3D Model Design 3.2 3D Printing 3.3 Surface Modification and Enzyme Immobilization on 3D Printed PLA Well-Plates 3.4 Enzymatic Activity Measurement in the PLA Well-Plates 4 Notes References Chapter 11: Rational Design of Self-Assembling Supramolecular Protein Nanostructures Utilizing the Cucurbit[8]Uril Macrocyclic... 1 Introduction 2 Materials 2.1 Addition of Phenylalanine-glycine-glycine (FGG-) to the N-terminal Site of Selected Enzyme Forms 2.2 Labeling of the Purified Enzyme with Fluorescein Isothiocyanate (FITC) 2.3 Addition of CB[8] to the FITC-Enzyme at Different Molar Ratios and Examination of Aggregate Formation Induced by CB[8] 3 Methods 3.1 Addition of Phenylalanine-glycine-glycine (FGG-) Tag at the N-terminal Site of Selected Enzyme Forms 3.2 Labeling of the Purified Enzyme with Fluorescein Isothiocyanate (FITC) 3.3 Addition of CB[8] to the FITC-Enzyme at Different Molar Ratios and Examination of Aggregate Formation Induced by CB[8] 4 Notes References Chapter 12: Lectin-Mediated Coimmobilization of Cascade Glycoenzymes 1 Introduction 2 Materials 3 Methods 3.1 Agglutination of GOx and HRP with ConA 3.2 Agglutination of GOx and HRP with ConA-Coated Magnetic Nanoparticles 3.2.1 Synthesis of Magnetic Nanoparticles 3.2.2 Preparation of ConA-Coated Magnetic Nanoparticles 3.2.3 Coimmobilization of GOx and HRP on ConA-Coated MNPs 3.2.4 Activity Assay 4 Notes References Chapter 13: Self-Assembled Multienzyme Nanostructures for Biocatalysis in Cellulo 1 Introduction 2 Materials 2.1 Bacterial Media 2.2 Plasmid Construction 2.3 Protein Expression 2.4 Product Extraction and Quantification 2.5 Transmission Electron Microscopy (TEM) 3 Methods 3.1 Plasmid Construction 3.2 Construction of Multienzyme Complexes in E. coli 3.3 Product Extraction and Quantification 3.4 Transmission Electron Microscopy (TEM) 4 Notes References Chapter 14: Nanoarmored Multi-Enzyme Cascade Catalysis 1 Introduction 2 Materials 2.1 Preparation of ZrP-BSA-Enzyme Suspension 2.2 Preparation of MWCNT/BSA Dispersion 2.3 Synthesis of BioCNT-GOx-HRP Nanomaterial Complex 2.4 Removing Unbound (Free) Enzymes and Proteins from the CNT Complex 2.5 Enzyme Activity Studies of Bio-nanomaterials Bound to Enzymes 3 Methods 3.1 Preparation of BioZrP-HRP-GOx Complex in a Single Step and Enzyme Nanoarmoring 3.1.1 Preparation of BioZrP-HRP-GOx Suspension 3.1.2 Binding of Enzymes to α-ZrP 3.1.3 TEM Characterization of Materials 3.1.4 Activity of GOx in GOx/Peroxidase Enzyme/BSA/ZrP Suspension 3.1.5 Activity of HRP in GOx/Peroxidase Enzyme/BSA/ZrP Suspension 3.2 Preparation of BioCNT/HRP/GOx Complex in One Step and Enzyme Nanoarmoring 3.2.1 Preparation of CNT-BSA Dispersion 3.2.2 Removal of Free BSA from CNT Dispersion 3.2.3 Preparation of BioCNT/HRP/GOx Complex 3.2.4 Removal of the Excess Enzymes 3.2.5 Binding and Activity of GOx to BioCNT-HRP-GOx 3.2.6 Binding and Activity of HRP in bioCNT-HRP-GOx 4 Notes References Chapter 15: Implementing Multi-Enzyme Biocatalytic Systems Using Nanoparticle Scaffolds 1 Introduction 2 Materials 2.1 Equipment 2.2 Reagents 2.2.1 Reagents for Bacterial Production of Enzymes 2.2.2 Reagents to Confirm Enzyme-QD Assembly via Gel Electrophoresis 2.2.3 Reagents for Enzyme Cascade Reactions 2.2.4 Reagents for LCMS Analysis 3 Methods 3.1 Nanoparticles 3.2 Protein Expression and Purification 3.2.1 Protein Expression 3.2.2 Cellular Lysis 3.2.3 Protein Purification 3.3 Confirming Assembly of Enzymes to NPs 3.3.1 Electrophoretic Mobility Shift Assay 3.3.2 Other Methods 3.4 Kinetic Assay for Observing Modified Flux with Enzyme-QD Conjugates 3.4.1 Multi-Enzyme Assembly onto QDs 3.4.2 Serial Dilution of Enzyme-QD Bioconjugates Experiment 3.5 Mass Spectral Analysis 4 Notes References Chapter 16: A Four-enzyme Nanoassembly Consisting of Hydrolases and Oxidoreductases for Multi-step Cascade Reactions 1 Introduction 2 Materials 2.1 Synthesis of Amino-functionalized Magnetic Nanoparticles (γ-Fe-Aptes-MNPs) 2.2 Co-immobilization of Cellulase, bgl, GOx, and HRP on γ-Fe-Aptes-MNPs 2.3 Determination of Enzyme Activity Recovery 2.4 Characterization of γ-Fe-Aptes-MNPs and γ-Fe-Aptes-MNPs-4enzymes - Fourier-transform Infrared Spectroscopy (FTIR) 2.5 Activity and Kinetic Studies of Free and Co-immobilized Enzymes 2.6 Monitoring the Reaction Progress of the 4-step Cascade Biotransformation of Cellulose 3 Methods 3.1 Synthesis of γ-Fe-Aptes-MNPs 3.2 Co-immobilization of Cellulase, bgl, GOx, and HRP on γ-Fe-Aptes-MNPs 3.3 Determination of Enzymes Activity Recovery 3.4 Characterization of γ-Fe-Aptes-MNPs the Nanobiocatalyst-FTIR 3.5 Activity and Kinetic Studies of Free and Co-immobilized Cellulase 3.6 Activity and Kinetic Studies of Free and Co-immobilized bgl 3.7 Activity and Kinetic Studies of Free and Co-immobilized GOx 3.8 Activity and Kinetic Studies of Free and Co-immobilized HRP 3.9 Monitoring the Reaction Progress of the 4-step Cascade Biotransformation of Cellulose 4 Notes References Chapter 17: A Bi-enzymatic Immobilized Nanobiocatalyst for the Biotransformation of Oleuropein to Hydroxytyrosol 1 Introduction 2 Materials 2.1 Synthesis of Chitosan-Coated Magnetic Nanoparticles (CS-MNPs) 2.2 Co-immobilization of bgl and CalA on Chitosan-Coated Magnetic Nanoparticles 2.3 Determination of Enzymes Activity Recovery 2.4 Characterization of Nanobiocatalysts-Field Emission Scanning Electron Microscopy (FE-SEM) 2.5 Characterization of Nanobiocatalysts: Circular Dichroism (CD) 2.6 Kinetic Studies of Free and Co-immobilized Enzymes 2.7 Reusability Studies of Bi-enzymatic Nanobiocatalyst 2.8 Monitoring the Reaction Progress: High-Pressure Liquid Chromatography (HPLC) 2.9 Liquid Chromatography-Mass Spectrometry Analysis (LC-MS) 3 Methods 3.1 Synthesis of Chitosan-Coated Magnetic Nanoparticles 3.2 Co-immobilization of bgl and CalA on Chitosan-Coated Magnetic Nanoparticles 3.3 Determination of Enzymes Activity Recovery 3.4 Characterization of CS-MNPs and CS-MNPs-2 Enzymes: Scanning Electron Microscopy (SEM) 3.5 Circular Dichroism Studies 3.6 Kinetic Studies of Free and Immobilized Biocatalysts 3.7 Re-use Studies of the Bi-enzymatic Nanobiocatalyst 3.8 Monitoring the Reaction Progress: HPLC 3.9 Liquid Chromatography-Mass Spectrometry Analysis (LC-MS) 4 Notes References Chapter 18: A Simple Recombinant E. coli Cell Lysate-Based Biocatalyst for ATP-Dependent Multi-step Reactions 1 Introduction 2 Materials 2.1 Growth of Recombinant Bacterial Cells 2.2 Preparation of Cell-Free Extract Biocatalyst 2.3 Cell-Free Extract Catalyzed Synthesis of ATP from AMP/ADP 2.4 HPLC Analysis of ATP Synthesis 2.5 Cell-Free Extract Catalyzed Synthesis of Citrulline Coupled to ATP Recycling 2.6 LC-TQD Analysis of Citrulline 3 Methods 3.1 Growth of Recombinant Bacterial Cells 3.2 Preparation of Cell-Free Extract 3.3 Cell-Free Extract Storage 3.4 Lyophilization 3.5 Assay for Cell-Free Extract Catalyzed Synthesis of ATP from AMP/ADP 3.6 HPLC Analysis of ATP Synthesis 3.7 Multi-enzymatic Synthesis of Citrulline 3.8 LC-TQD Analysis of Citrulline 4 Notes References Chapter 19: CO2 to Methanol: A Highly Efficient Enzyme Cascade 1 Introduction 2 Enzymes Involved in the Multi-enzymatic CO2 Reduction to Methanol 2.1 Formate Dehydrogenase (FDH) 2.2 Formaldehyde Dehydrogenase (FaldDH) 2.3 Alcohol Dehydrogenase (ADH) 3 Immobilization as a Strategy to Develop Robust Multi-enzymatic CO2 Reducing Systems 3.1 Co-immobilization 3.2 Sequential Immobilization 4 Addressing the Need for Cofactor Regeneration 5 Integrated Approaches for CO2 Reduction to Methanol with In Situ Cofactor Regeneration 5.1 Enzyme Catalysis 5.2 Enzymatic Photocatalysis 5.3 Enzymatic Electrocatalysis 5.4 Enzymatic Photoelectrocatalysis 6 Addressing CO2 Solubilization as a Limiting Factor 6.1 Use of Carbonic Anhydrase 6.2 Use of Co-solvents 7 Conclusions References Chapter 20: Multienzyme Catalysis in Phase-Separated Protein Condensates 1 Introduction 2 Materials 2.1 Recombinant Proteins Expression and Purification 2.2 Phase Separation, Confocal Imaging, and Centrifugation/SDS-PAGE Assay 2.3 Enzymatic Reaction of Terpene Biosynthesis Enzymes 3 Methods 3.1 Cloning, Recombinant Protein Expression, and Purification 3.1.1 Protein Expression 3.1.2 Protein Purification 3.2 Fluorescent Protein Labeling 3.3 Phase Separation and Confocal Imaging Assay 3.4 Analyze Protein Distribution in Condensate by Centrifugation/SDS-PAGE Assay 3.5 Enzymatic Reaction of Terpene Biosynthesis Inside Phase Condensates 4 Notes References Chapter 21: Robust and Continuous Lipase-Catalyzed Reactions in Deep Eutectic Solvents: Low Viscosity and Double CLEA-LentiKat... 1 Introduction 2 Materials 2.1 Reagents 2.2 Enzymes 2.3 Analytical Equipment 2.4 Equipment 3 Methods 3.1 DES Preparation and Lipase-Catalyzed Esterification (See also Ref.) 3.2 Cross-Linking Aggregates Immobilization of Lipases (CLEA-Lipase) 3.3 Immobilization in LentiKats 3.4 Set-up of a Continuous Process with CLEA-LK-lipases and Low Viscous DES-Buffer Media 4 Notes References Chapter 22: Enzymatic Photometric Assays for the Selective Detection of Halides 1 Introduction 1.1 Selective Assays 2 Materials 2.1 Peroxidases 2.2 Cultivation and Purification of CiVCPO and the Triple Mutant 2.3 Halide Assay Reagents 2.4 Halide Standard Solutions 2.5 Reaction Mixtures for the Assays 2.6 Consumables and Measuring Devices 3 Methods 3.1 Cultivation and Purification of CiVCPO and the Triple Mutant 3.2 Preparation of CoVBPO 3.3 Determination of Haloperoxidase Activities 3.4 General Halide Assay (HOX Assay) 3.5 Selective Bromide Detection in the Presence of Chloride 3.6 Iodide Detection 4 Notes References Index