Methods in Enzymology

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Contributors
Preface
Current status and future development of plastics: Solutions for a circular economy and limitations of envir ...
	Introduction
	Definitions
		Polymer or plastic-What is the difference?
		What is bio-based?
	Biodegradation and biodegradability-Explanations and limits
		Certified biodegradable polymers and derived plastics
	Current plastic market and production
		Fossil-based plastics
		Bio-based plastics
		Biodegradable plastics
		CO2-based plastics
	Plastic recycling
	Plastic waste and disposal
	Mismanagement of plastic disposal and recycling-Plastic littering
	Political actions against plastic littering
	Conclusion and future development
	Methods
	References
Methods for microplastic sampling and analysis in the seawater and fresh water environment
	Introduction
	Water sample collection methods
		Towing
			Materials
			Sample collection procedures
		Direct pumping
			Materials
			Sample collection procedure
		Bulk sampling
			Materials
			Sample collection procedure
	Sample pretreatment before identification
		Materials
		Sample pre-treatment procedure
	Sample identification
		Visual inspection with a stereomicroscope
		Microplastic polymer composition identification
	Data reporting
	Blank and contamination control
	Summary
	Acknowledgments
	References
Exploring microbial consortia from various environments for plastic degradation
	Introduction
	Enrichment and cultivation of aerobic plastic-degrading consortia
		Soil and compost
		Freshwater and seawater
			Protocol for the enrichment of marine aerobic consortia
				Materials, equipment and reagents
					Media
					Equipment
					Chemicals
				Protocol
		Methods to quantify plastic aerobic mineralization
	Enrichment and cultivation of anaerobic plastic-degrading consortia
		Soil and landfills
		Anaerobic digesters and fermentation tanks
		Marine ecosystem
			Protocol for the enrichment of marine anaerobic consortia
				Materials, equipment and reagents
					Media
					Equipment
				Protocol
					Media Preparation
					Sample Collection and Preparation
		Methods to quantify plastic anaerobic mineralization
	Future perspectives
	References
Cultivation of filamentous fungi for attack on synthetic polymers via biological Fenton chemistry
	Introduction
		Types and biodegradability of plastics
		Extracellular Fenton-type reactions in filamentous fungi
	Rationale for the cultivation of fungi for Fenton chemistry-dependent attack on synthetic polymers
	Materials, equipment and reagents
		Materials
		Equipment
		Chemicals
	Protocols
		Maintenance of fungal cultures
		Preparation of water-soluble PSS for depolymerization experiments
		Preparation of solid polymer films for degradation experiments
		Depolymerization of water-soluble PSS using brown-rot basidiomycetes
		Depolymerization of water-soluble PSS using white-rot basidiomycetes and filamentous ascomycetes
		Investigating biochemical attack on solid polymer films by brown-rot basidiomycetes
		Investigating biochemical attack on solid polymer films by white-rot basidiomycetes and filamentous ascomycetes
	Safety considerations
	Applicable analytical methods to detect fungal effects on polymers
		Size exclusion chromatography (SEC) for the analysis of water-soluble polymers
		Water contact angle analysis of solid polymer surfaces
		X-ray photoelectron spectroscopy (XPS) analyses of solid polymer surfaces
	Summary
	Acknowledgments
	References
Characterization of biodegradation of plastics in insect larvae
	Introduction
	Biodegradation of plastics in Tenebrio molitor
		Incubation conditions for biodegradation of plastics
			Plastic feedstock
			Effect of co-diet
			Temperature and humidity
			Rearing density
		Gut microbes
		Enrichment and isolation of plastic-degrading gut microbes
	Analytical methods for plastic biodegradation
		Gravimetric determination of weight loss
		Chemical and structure modification
			Gel permeation chromatography (GPC)
			Fourier transform infrared spectroscopy (FTIR)
			Thermogravimetrical analysis (TGA)
			Proton nuclear magnetic resonance (H NMR)
			Differential scanning calorimetric (DSC) analysis
		Surface observation
			Microscopic observations
			Water contact angle (WCA)
		Isotopic labeling and other methods
			Isotopic labeling
			CO2 and methane production-based biodegradation methods
			Other methods
	Protocols for the characterization of plastic degradation by T. molitor larvae
		Equipment, supplies and chemicals
			Equipment and supplies
			Chemicals
		Operational protocols
			Feedstock preparation
			Set up of incubators with plastic foam as target feedstock
			Set up of incubators with microplastics (MPs) as feedstock
			Larval counting and frass collection
			Sampling for gut microbiome analysis
			Calculation of physiological parameters of the larvae
			Residual fractions in frass
			Polymer (PS) removal
			GPC analysis
			FTIR Analysis
			TGA analysis
			H NMR analysis
			Water contact angle (WCA) analysis
			Gut Microbial enrichment and bacterial isolation
	Results and analysis
		Larval activities and degradation rates
			Survival rates and PS consumption of mealworms
		Characterization of biodegradation
			THF extractable fraction in frass
			GPC analysis
			FTIR analysis of frass
			H NMR analysis
			TGA analysis
		Plastic-degraders
	Summary
	Acknowledgments
	References
Quantification of polystyrene plastics degradation using C isotope tracer technique
	Introduction
	Protocols
		Synthesis of [β-C]-styrene
			Materials
			Equipment
			Method
				Synthesis of [β-C]-styrene
				Synthesis of [U-ring-C]-styrene
		Synthesis of C-PS film
			Materials
			Equipment
			Method
		Synthesis of C-PS nanoplastics
			Materials
			Equipment
			Method
		Biodegradation of C-PS film by Penicillium variabile
			Materials
			Equipment
			Method
		Photodegradation of [β-C]-PS nanoplastics
			Materials
			Equipment
			Method
		LSC analyses for C in liquid phases
			Materials
			Equipment
			Method
		C-plastics
			Materials
			Equipment
			Method
		C-HPLC analyses for degradation products in liquid phase
			Materials
			Equipment
			Method
	Summary and outlook
	Safety notes
	Acknowledgments
	References
Exploring the global metagenome for plastic-degrading enzymes
	Introduction
		Plastics as environmental pollutants
		Degradation processes of plastic
			Biotic disruption of PET
			PETases: PET-degrading α/β-hydrolases
		Metagenome screening for novel PETases
	Materials
	Methods
		Construction of a profile hidden Markov model (HMM) and screening of a protein database for putative PETases
		Functional screening on polymer-containing indicator plates
			PET-containing plates
			PCL-containing plates
			BHET-containing plates
	Notes
	Outlook and summary
	References
Cutinases from thermophilic bacteria (actinomycetes): From identification to functional and structural cha ...
	Introduction
	Identification of thermophilic cutinases
		Identification and cloning of thermophilic cutinases from actinomycetes
		Purification and characterization of polyester-hydrolyzing cutinases
	Structural and thermodynamic analysis of PET-hydrolyzing cutinases
		Crystal structures of thermophilic cutinases
		Structural dynamics driven by Ca-binding and complexation with model substrates
		Thermodynamic analysis of metal binding and its effects on stability
	Protocols
		Cloning of cutinases
			Equipment
			Materials
			Procedure
		Purification of cutinases
			Equipment
			Materials
			Procedure
		Activity assay of cutinases
			Equipment
			Chemicals
			Procedure
		Crystallization and crystal structure analysis
			Equipment
			Materials
			Procedure
		MD simulations
			Materials
			Procedure
		ITC analysis
			Equipment
			Chemicals
			Procedure
		CD analysis
			Equipment
			Chemicals
			Procedure
		DSC analysis
			Equipment
			Chemicals
			Procedure
	Summary
	Acknowledgments
	References
Ideonella sakaiensis, PETase, and MHETase: From identification of microbial PET degradation to enzyme chara ...
	Introduction
	Screening microorganisms that degrade PET and isolation of microbial consortium no. 46
		Materials
		Procedure
	Isolation of I. sakaiensis201-F6 from microbial consortium no. 46
		Materials
		Procedure
	Detection of microbial PET degradation
		Weight loss
			Procedure
		CO2 generation
			Equipment
			Procedure
		SEM
			Equipment
			Procedure
		XPS
			Equipment
			Chemicals
			Procedure
	Characterization of PETase and MHETase
		Cloning, expression, and purification of PETase and MHETase
		Characterization of PETase and other PHEs
			PET (solid material) degradation assay
				Equipment
				Procedure
			Assay of enzymatic BHET hydrolysis
				Procedure
			Plate reader assays for para-nitrophenol (pNP)-aliphatic esters
				Chemicals and reagents
				Procedure
			Temperature-dependence assay
				Procedure
		Characterization of MHETase
			Enzymatic preparation of MHET crude solution
			Kinetic analysis of MHETase
	Summary
	Acknowledgments
	Declarations of interest
	References
GRAPE, a greedy accumulated strategy for computational protein engineering
	Introduction
	Materials
	Methods
		Setting up working directories and preparing target structure
		DeltaDeltaG calculation with FoldX
		DeltaDeltaG calculation using Rosetta
		ABACUS energy calculation by ABACUS and filtering
		Consensus analysis
		Selecting computationally designed and consensus analysis yielded variants for wet-lab characterization
		Experimental verification of the filtered mutations
		K-means clustering of mutations with significant improvement of protein melting temperature
		Greedy accumulation of beneficial mutations to an ultimate hyper-stable mutant
		Further characterization of the mutants
	Notes
	Summary
	References
Mechanistic investigation of enzymatic degradation of polyethylene terephthalate by nuclear magnetic reso ...
	Introduction
	H solution NMR analysis to quantify PET chain scissions
		Protocol
			Equipment
			Chemicals
			Preparation
			Data evaluation
	Solid-state NMR analysis to determine PET chain conformation and dynamics
		Protocol
			Equipment
			Chemicals
			Preparation
			Data evaluation
		Cross polarization for determination of PET chain conformation
			Data acquisition of the CP experiments
			Data acquisition and processing of the HETCOR experiments
		Dynamics investigations
			Rotating-frame spin-lattice relaxation to investigate PET chain dynamics
				Data acquisition and evaluation of the T1ρ experiments
			Separation of undistorted powder patterns by effortless recoupling to study reorientations of the carbonyl group i ...
				Procedure to determine the orientation of principal axes for PET carbonyl carbon by DFT-based calculations
				Data acquisition and evaluation of the SUPER experiments
			Dipolar chemical shift correlation to quantify localized dynamics
				Data acquisition and evaluation of the DIPSHIFT experiments
			Centerband-only detection of exchange to characterize reorientations of the PET phenylene unit
				Data acquisition and evaluation of the CODEX experiments
			Exchange spectroscopy for investigation of gauche/trans conformational exchange in PET
	Summary
	Acknowledgments
	Funding
	References
Fluorimetric high-throughput screening method for polyester hydrolase activity using polyethylene terepht ...
	Introduction
	PET nanoparticles: Generation, application, and characterization
	Fluorimetric high-throughput screening assay
	Materials, equipment, and reagents
		PET NP production
		PET NP characterization
		Protein production
		Enzymatic degradation of PET NPs
		Determination of polyester hydrolysis activity
			Fluorimetric HTS assay
			RP-HPLC
	Protocols
		PET NP production
		PET NP characterization
			Analysis of particle size and morphology by SEM
			Analysis of particle size distribution by DLS
		Protein production
		Enzymatic degradation of PET NP
		Quantification of the PET degradation products
			Fluorimetric HTS assay
			RP-HPLC
	Acknowledgments
	References
Anchor peptides promote degradation of mixed plastics for recycling
	Introduction
	Engineering the adhesion peptide binding modules for enhanced polymer absorption and enzymatic degradation
	Equipment
	Materials
	Protocol
		Generation of EGFP-anchor peptide fusion constructs
		Expression of EGFP-anchor peptide fusion proteins
		Purification of EGFP-anchor peptide fusion proteins
		Anchor peptide binding assay
		Directed evolution of the anchor peptide for improved binding affinity
		Production of enzyme-anchor peptide fusion protein
		Plastic degradation promoted by anchor peptides
	Summary and outlook
	Acknowledgments
	References
Tuning of adsorption of enzymes to polymer
	Introduction
	Expression and purification of polymer degrading hydrolases
		Materials and reagents
		Buffers
		Strains
		Equipment
		Protocols
	Surface engineering by site-directed mutagenesis
		Materials and reagents
		Buffers
		Equipment
		Protocols
	Fusion of hydrophobic binding domains
		Software
		Protocols
		Application of hydrophobins
		Materials and reagents
		Strains
		Protocols
	Enzyme truncation and mutagenesis of the metal-binding site
		Materials and reagents
		Strains
		Equipment
		Protocols
	Summary
	References
Characterization of the enzymatic degradation of polyurethanes
	Introduction
		PU structures and applications
		Mechanism of enzymatic PUR degradation
	Materials, equipment and reagents
		Equipment
		Materials
		Reagents
	Protocol
		Polymer synthesis
		Polymer degradation
		Polymer analysis
			Weight loss measurement
			Scanning electron microscopy (SEM)
			Size exclusion chromatography (SEC)
			Fourier-transform infrared spectroscopy (FTIR)
			Thermal analysis
		Degradation product analysis
			H NMR)
			Liquid chromatography-Mass spectrometry (LC-MS)
	Safety considerations and standards
	Analysis and statistics
	Pros and cons
	Alternative methods/procedures
	Troubleshooting and optimization
	Summary
	Acknowledgments
	References
Structural analysis of PET-degrading enzymes PETase and MHETase from Ideonella sakaiensis
	Introduction
	Methods background
		Expression and purification
		Crystallization
		Modeling
	Expression and purification
		Equipment
		Chemicals
		Protocol
			Expression of PETase
			Purification of PETase
			Expression of MHETase
			Purification of MHETase
	Crystallization
		Equipment
		Chemicals
		Protocol
			Crystallization of PETase
			Crystallization of MHETase
		Successful crystallization conditions
	Modeling of a PETase-BHET complex
		Protocol
			Protocol for in silico docking with SwissDock
			Protocol for in silico docking with Autodock Vina
	Summary
	Acknowledgments
	References
Structural and functional characterization of nylon hydrolases
	Introduction
	Screening of microorganisms and enzymes degrading nylon-related compounds
	Preparation of oligomeric and polymeric substrates for enzyme assays
		Preparation of 6-aminohexanoate oligomers
		Limited hydrolysis of nylons by formic acid
		Interfacial polymerization of nylon-66, nylon-66/nylon-64 copolymer
	Enzymatic hydrolysis of nylons and related substrates
		Enzyme assay for nylon oligomers
		Enzyme assay for powdered nylons
		Enzyme assay for thin-layered nylons
	Structural analysis of 6-aminohexanoate dimer hydrolase NylB
		Crystallization
		Crystallographic analysis of substrate-unbound enzyme
		Crystallographic analysis of enzyme-substrate complex
		Computational analysis
	Protocol
		Screening of microorganisms degrading nylon-related compounds
		Chemical synthesis of 6-aminohexanoate linear dimer
		Limited hydrolysis of polymeric nylons by formic acid
		Interfacial polymerization of nylon-66/nylon-64 copolymer
		Gel permeation chromatography (GPC) analyses of nylons
		Qualitative detection of the enzyme activity by thin-layer chromatography (TLC)
		Quantitative analysis of reaction products by high-performance liquid chromatography (HPLC)
		Quantification of amino groups by a colorimetric method
		Enzymatic degradation assay with nylon powder
		Preparation of thin-layered nylons
		Enzymatic hydrolysis of thin-layered nylons
		Estimation nylon film thickness by imaging analysis
		Crystallization of NylB
		Collection of diffraction data in X-ray crystallographic analysis
		Phase determination, model building and crystallographic refinement
		X-ray crystallographic analysis of enzyme-substrate complex
		Molecular dynamic (MD) analysis
		Quantum mechanics/molecular mechanics (QM/MM) analysis
	Summary
	Acknowledgments
	References
Upcycling of hydrolyzed PET by microbial conversion to a fatty acid derivative
	Introduction
	Protocols
		Adaptive laboratory evolution (ALE)
			Materials, equipment, and reagents
				Materials
				Equipment
				Reagents
			Method
				Cultivation
				Genome (re-)sequencing
				Reverse engineering
			Alternative procedures
		Determination of substrate consumption: EG quantification
			Materials, equipment, and reagents
				Material
				Equipment
				Reagents
			Method
		Determination of substrate consumption: TA quantification
			Materials, equipment, and reagents
				Material
				Equipment
				Reagents
			Method
		Genetic engineering for HAA synthesis
			Materials, equipment, and reagents
				Strain and plasmid
				Equipment
				Reagents
			Method
				Preparation of electrocompetent cells
				Electroporation of electrocompetent Pseudomonas cells
		Biosynthesis of HAA
			Materials, equipment, and reagents
				Strain
				Equipment
				Reagents
			Method
		Quantification of HAA
			Materials, equipment, and reagents
				Material
				Equipment
				Reagents
			Method
			Alternative procedure
		HAA purification
			Materials, equipment, and reagents
				Material
				Equipment
				Reagents
			Method
				Separation of HAA from the supernatant
				Purification of HAA by preparative HPLC
	Conclusion
	Acknowledgments
	References
Screening and cultivating microbial strains able to grow on building blocks of polyurethane
	Introduction
	Methods
		Isolation and characterization of bacteria from soil samples capable of degrading PU building blocks
			Rationale
			Materials, equipment and reagents
			Protocol
			Safety considerations and standards
			Analysis and statistics
			Alternative methods/procedures
		Quantification of TDA degradation
			Rationale
			Materials, equipment and reagents
			Protocol
			Safety considerations and standards
			Analysis and statistics
		Quantification of PU diol degradation
			Rationale
			Materials, equipment and reagents
			Protocol
			Analysis and statistics
			Alternative method
	Outlook
	Acknowledgment
	References
Engineering microalgae as a whole cell catalyst for PET degradation
	Introduction
	Microalgae as phototrophic cell factories for recombinant protein production
		Design of the genetic constructs
		Genomic transformation
		Screening and clone selection
		Determining optimal expression conditions
		Plastic degradation assays
	Introducing the PETase gene into the genome of a diatom
		Equipment
		Chemicals
	Protocol
		Generating the genetic construct for transformation
		Transformation of the diatom
		Testing the expression and secretion of the recombinant PET hydrolase
		Analyzing the functionality of the PETase against its substrate
		PET degradation experiments
	Analysis of enzymatic plastic degradation
		Scanning electron microscopy
		High performance liquid chromatography
	Summary
	Acknowledgments
	References
Yeast cell surface display of bacterial PET hydrolase as a sustainable biocatalyst for the degradatio ...
	Introduction
		The current status of enzymatic PET depolymerization
		IsPETase as a promising biocatalyst for PET depolymerization
		Brief introduction of whole-cell biocatalysts
		Brief introduction of the yeast surface display system
		Application of whole-cell biocatalyst in lipase and cutinase
	Protocols
		Construction of a whole-cell biocatalyst by displaying IsPETase on the surface of yeast (Pichia pastoris) cells
		Verification of IsPETase expression
			Western Blot
			Immunofluorescence microscopy analysis
		Detection of release products by high-performance liquid chromatography (HPLC)
		Optimization of the surface display system
			Materials
		The application of the whole-cell biocatalyst
			Materials
	Acknowledgment
	References