Recombinant Gloeophyllum trabeum Endoglucanase Cel5A

What is Gloeophyllum trabeum Cel5A and what makes it significant in cellulose degradation research?

Gloeophyllum trabeum Cel5A is a 42-kDa endoglucanase produced by the brown rot fungus Gloeophyllum trabeum. It holds particular significance in cellulose degradation research because it exhibits processive activity on crystalline cellulose, specifically microcrystalline cellulose (Avicel). Unlike many other endoglucanases from brown rot fungi, G. trabeum Cel5A demonstrates significant Avicelase activity, measured at 4.5 nmol glucose equivalents released per minute per mg protein .

The enzyme is noteworthy because it hydrolyzes Avicel primarily to cellobiose while introducing only a small proportion of reducing sugars into the remaining insoluble substrate, indicating its processive mechanism of action . This characteristic makes G. trabeum Cel5A valuable for understanding enzymatic degradation of recalcitrant cellulosic materials in natural settings and potential biotechnological applications.

How does G. trabeum Cel5A compare with other endoglucanases structurally and functionally?

While the search results don't provide specific structural information about G. trabeum Cel5A, we can make comparative inferences based on information about related cellulases. Structurally, endoglucanases in the GH5 family (including Cel5A enzymes) typically exhibit a (β/α)8 TIM-barrel fold, as seen in Cel5A from Hypocrea jecorina and Thermotoga maritima .

Functionally, G. trabeum Cel5A differs from many other brown rot fungal endoglucanases in its ability to degrade crystalline cellulose. Previous reports indicated that other endoglucanases from G. trabeum were not capable of degrading crystalline cellulose . When compared to industrial cellulases like those from Hypocrea jecorina (Trichoderma reesei), G. trabeum Cel5A represents part of a different cellulose degradation strategy. While H. jecorina employs a comprehensive suite of cellulases, G. trabeum combines enzymatic activity with a non-enzymatic component - a hydroquinone-driven system for the production of extracellular reactive oxygen species (ROS) .

What expression systems are suitable for producing recombinant G. trabeum Cel5A?

While the search results don't specifically address expression systems for G. trabeum Cel5A, we can extrapolate from methods used for similar enzymes. For thermophilic and mesophilic cellulases, Escherichia coli expression systems have been successfully employed, as demonstrated with Cel5A from Thermotoga maritima .

The methodology would typically involve:

• Gene cloning: Isolation of the cel5A gene from G. trabeum and cloning into a suitable expression vector

• Expression optimization: Testing various E. coli strains, such as BL21(DE3) derivatives

• Induction protocols: Implementing either IPTG induction or autoinduction methods

• Temperature optimization: Using lower temperatures (20-30°C) during the induction phase to improve soluble protein yields

• Protein extraction: Using methods like PopCulture reagent for high-throughput applications or conventional cell lysis for larger scale production

For improved expression, a two-stage protocol with growth at 37°C followed by protein expression at 30°C may be beneficial, as this approach has proven successful with other cellulases .

What are the standard assays for measuring G. trabeum Cel5A activity?

Based on the methodologies described for similar cellulases, several standard assays can be used to measure G. trabeum Cel5A activity:

• DNS (3,5-dinitrosalicylic acid) reducing sugar assay: This colorimetric method measures the release of reducing sugars from cellulosic substrates. The assay can be performed using DNS reagent with or without sulfite and phenol, with the former offering better sensitivity but the latter being more environmentally friendly .

• Activity on soluble substrates: Carboxymethyl cellulose (CMC) is commonly used as a substrate for measuring endoglucanase activity. The activity can be quantified through the DNS assay or using Congo red staining to visualize zones of hydrolysis on agar plates .

• Activity on crystalline cellulose: Given Cel5A's ability to degrade Avicel, activity can be measured by quantifying the release of cellobiose and other sugars from microcrystalline cellulose. This can be done using high-performance liquid chromatography (HPLC) or specific enzymatic assays for cellobiose .

• Processivity measurement: The ratio of soluble reducing sugars released to reducing ends created on the insoluble substrate can be used to assess the processivity of the enzyme .

How does the processive mechanism of G. trabeum Cel5A differ from classical cellobiohydrolases, and what structural features enable this processivity?

G. trabeum Cel5A exhibits a unique processive mechanism that distinguishes it from classical cellobiohydrolases while retaining its classification as an endoglucanase. Unlike typical cellobiohydrolases that initiate hydrolysis from chain ends, G. trabeum Cel5A can make initial cuts within the cellulose chain (endo-activity) but then proceeds to slide along the chain releasing cellobiose units without dissociating from the substrate (processive activity) .

• A tunnel-like active site architecture that partially encloses the cellulose chain

• Surface-exposed aromatic residues that facilitate binding to crystalline cellulose

• Specific loop regions that enable the enzyme to remain associated with the substrate after catalysis

Research methodologies to investigate these structural features would include:

• X-ray crystallography of G. trabeum Cel5A, ideally in complex with oligosaccharide substrates

• Site-directed mutagenesis of potential processivity-determining residues

• Molecular dynamics simulations to understand substrate binding and enzyme movement

What role does G. trabeum Cel5A play in the context of the complete lignocellulose degradation system of brown rot fungi?

G. trabeum Cel5A functions as a key component within the broader lignocellulose degradation system of brown rot fungi. Unlike white rot fungi that employ an extensive array of ligninolytic enzymes, G. trabeum and other brown rot fungi have evolved a more streamlined approach combining limited enzymatic capabilities with non-enzymatic mechanisms.

The research indicates that G. trabeum produces:

• A hydroquinone-driven system for generating extracellular reactive oxygen species (ROS)

• A β-glucosidase

• A xylanase (Xyn10A)

• At least three cellulases: the 42-kDa Cel5A, the 28-kDa Cel12A, and other endoglucanases

With the discovery of Cel5A's activity on crystalline cellulose, it's now clear that G. trabeum possesses "all of the components currently thought necessary for wood decay" . The proposed mechanism involves:

• Initial attack by ROS that creates access points in the lignocellulose matrix

• Cel5A action on exposed crystalline cellulose regions, generating primarily cellobiose

• Potential synergistic action with Cel12A on more accessible regions

• Conversion of cellobiose to glucose by β-glucosidase for fungal metabolism

• Simultaneous degradation of hemicellulose by Xyn10A

To comprehensively study this system, researchers should employ techniques including:

• Transcriptomics and proteomics under different growth conditions

• Enzyme purification and characterization of individual components

• Reconstitution experiments to determine minimal components required for effective degradation

• Imaging techniques to visualize the progression of decay in wood samples

What strategies can be employed to improve the thermostability and specific activity of recombinant G. trabeum Cel5A through protein engineering?

While the search results don't provide specific information about engineering G. trabeum Cel5A, insights can be drawn from successful approaches with other thermophilic cellulases, particularly Cel5A from Thermotoga maritima (Cel5A_Tma).

Engineering strategies may include:

• Directed evolution approaches:

  • Error-prone PCR with an optimal mutation rate (approximately 4.8 bp/kb has been successful for Cel5A_Tma)

  • High-throughput screening assays at elevated temperatures (e.g., 70°C) to identify variants with improved thermostability and activity

  • Sequential screening on different substrates, beginning with soluble substrates (CMC) and progressing to industrially relevant lignocellulosic materials

• Rational design approaches:

  • Introduction of disulfide bridges to stabilize the protein structure

  • Surface charge optimization to enhance thermostability

  • Targeting residues distal to the active site that influence dynamic modes associated with activity

  • Modification of loop regions that may be involved in substrate binding or product release

• Structural considerations:

  • Focus on modifications to surface residues, as these have shown promise in improving cellulase activity without disrupting the catalytic core

  • Analysis of secondary structure elements to identify non-essential regions that can be modified to improve stability

  • Examination of the substrate binding groove to enhance substrate affinity

Based on findings with Cel5A_Tma, mutations located in loop regions and on the molecular surface at positions distal to the active site have resulted in activity improvements of 25-42% on CMC and 13-30% on pretreated biomass . This suggests that activity-enhancing mutations need not be limited to the active site and may work by "enhancing the coordination of dynamic modes associated with activity or by biasing the folding landscape toward the most active conformational state" .

How can synergistic interactions between G. trabeum Cel5A and other enzymes or non-enzymatic components be optimized for enhanced biomass degradation?

Optimizing synergistic interactions for enhanced biomass degradation requires understanding the natural synergies in the G. trabeum system and designing experimental approaches to leverage these interactions.

Research approaches should include:

• Characterization of natural synergies:

  • Investigation of interactions between Cel5A, Cel12A, and Xyn10A from G. trabeum

  • Analysis of how the hydroquinone-driven ROS system enhances cellulase accessibility to crystalline cellulose

  • Determination of optimal enzyme ratios in reconstituted systems

• Cross-species synergies:

  • Testing G. trabeum Cel5A in combination with commercial cellulase cocktails (e.g., from T. reesei)

  • Evaluation of synergies with auxiliary enzymes such as lytic polysaccharide monooxygenases (LPMOs) that use oxidative mechanisms to cleave cellulose chains

  • Combination with hemicellulases from various sources to address the heterogeneous nature of lignocellulosic biomass

• Non-enzymatic enhancement strategies:

  • Mimicking the natural Fenton chemistry of brown rot fungi with controlled application of hydrogen peroxide and iron compounds

  • Pretreatment approaches that mimic or complement the natural action of G. trabeum's ROS system

  • Investigation of surfactants or small molecules that might enhance Cel5A binding or activity

• Methodological considerations:

  • Development of standardized assays to accurately measure synergistic effects

  • Design of experiments that distinguish between true synergy and additive effects

  • Time-course studies to understand the optimal sequence of enzyme/component addition

What analytical techniques are most effective for characterizing the mode of action and processivity of G. trabeum Cel5A on different cellulosic substrates?

Characterizing the mode of action and processivity of G. trabeum Cel5A requires sophisticated analytical techniques that can provide insights into enzyme kinetics, substrate binding, and product profiles.

Recommended analytical techniques include:

• Product profiling techniques:

  • High-Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) to analyze the distribution of soluble oligosaccharides produced

  • Mass spectrometry to identify and quantify released products

  • Reducing end assays to determine the ratio of soluble reducing sugars to reducing ends created on the insoluble substrate (a measure of processivity)

• Substrate analysis techniques:

  • X-ray diffraction (XRD) to assess changes in cellulose crystallinity before and after enzyme treatment

  • Scanning electron microscopy (SEM) and atomic force microscopy (AFM) to visualize physical changes to substrate morphology

  • Fourier transform infrared spectroscopy (FTIR) to detect chemical modifications to the substrate

• Enzyme-substrate interaction studies:

  • Surface plasmon resonance (SPR) or quartz crystal microbalance with dissipation monitoring (QCM-D) to measure binding affinities and association/dissociation kinetics

  • Fluorescently labeled enzyme studies to track enzyme movement on cellulose surfaces

  • Isothermal titration calorimetry (ITC) to determine thermodynamic parameters of binding

• Structural studies:

  • X-ray crystallography of enzyme-substrate complexes to visualize binding modes

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify regions involved in substrate binding

  • Small-angle X-ray scattering (SAXS) to study conformational changes upon substrate binding

• Computational approaches:

  • Molecular dynamics simulations to model enzyme-substrate interactions and the processive mechanism

  • Quantum mechanics/molecular mechanics (QM/MM) calculations to understand the catalytic mechanism

  • Bioinformatic analyses to compare G. trabeum Cel5A with other processive and non-processive enzymes

The combination of these techniques would provide a comprehensive understanding of how G. trabeum Cel5A interacts with and degrades different cellulosic substrates, guiding both fundamental research and applications in biomass conversion.

What are the critical parameters for optimizing heterologous expression of recombinant G. trabeum Cel5A?

Successful heterologous expression of recombinant G. trabeum Cel5A requires careful optimization of several critical parameters:

• Host selection and strain engineering:

  • E. coli strains such as BL21(DE3) derivatives are commonly used for cellulase expression, with Acella strain (with endA and recA deletions) being particularly suitable for high-throughput screening

  • Consider specialized strains with enhanced disulfide bond formation capabilities if G. trabeum Cel5A contains disulfide bridges

  • For improved folding, strains co-expressing chaperones may be beneficial

• Vector design:

  • Optimize codon usage for the expression host

  • Select appropriate promoters (T7 for E. coli is common)

  • Consider fusion tags that may enhance solubility (e.g., SUMO, MBP) with appropriate cleavage sites

• Expression conditions:

  • Temperature: Lower temperatures (20-30°C) generally favor proper folding of recombinant cellulases

  • Implement a two-stage protocol: 37°C for cell growth followed by 30°C for protein expression

  • Induction method: Compare IPTG induction versus autoinduction media, which has shown good results for cellulase expression

  • Media composition: Rich media like 2xYT often yields better results for cellulase expression

• Protein extraction:

  • PopCulture reagent for high-throughput applications or conventional cell lysis for larger scale production

  • Evaluate the efficiency of different lysis methods (sonication, French press, enzymatic lysis)

  • Optimize buffer conditions to maintain enzyme stability during extraction

• Protein purification:

  • Design purification strategy based on fusion tags or native properties

  • Consider temperature-based purification steps if the enzyme is thermostable

  • Evaluate different buffer compositions for maximum stability and activity

How can researchers troubleshoot common challenges in activity assays for G. trabeum Cel5A?

Researchers working with G. trabeum Cel5A may encounter several challenges when conducting activity assays. Here are troubleshooting approaches for common issues:

• Low or inconsistent enzyme activity:

  • Verify enzyme concentration using standard protein quantification methods

  • Check buffer composition and pH; optimal conditions for Cel5A enzymes typically include slightly acidic pH and presence of divalent cations

  • Ensure substrate quality and consistent preparation (particularly important for insoluble substrates like Avicel)

  • Validate assay temperature control, as temperature fluctuations can significantly affect enzyme kinetics

  • Consider adding BSA or other stabilizing agents to prevent enzyme adsorption to surfaces

• Interference in reducing sugar assays:

  • If using DNS assay, test different formulations (with/without sulfite and phenol) to balance sensitivity and ease of disposal

  • Run appropriate blanks including substrate-only and enzyme-only controls

  • For complex biomass substrates, implement additional washing steps or use alternative detection methods

• Evaporation during high-temperature assays:

  • Add light mineral oil to prevent evaporation during both enzymatic and DNS colorimetric reactions

  • Use seal films or plate sealers designed for high-temperature applications

  • Consider using PCR tubes or plates for smaller volume, high-temperature reactions

• Poor reproducibility in processivity measurements:

  • Standardize substrate loading and mixing conditions

  • Implement precise timing protocols for sampling

  • Use internal standards for quantification

  • Consider specialized assays designed specifically for processivity measurement

• Challenges with high-throughput screening:

  • Optimize liquid handling parameters for consistency across all wells

  • Implement quality control measures such as including wild-type controls in each plate

  • Use statistical methods to identify and exclude outliers

  • Confirm hits with secondary validation assays

What experimental designs are most effective for studying the synergistic action of G. trabeum Cel5A with other components of the fungal degradation system?

Effective experimental designs for studying synergistic action should be systematic and control for multiple variables. Here are recommended approaches:

• Factorial experimental designs:

  • Design experiments that test multiple enzyme combinations at various ratios

  • Include G. trabeum Cel5A, Cel12A, Xyn10A, and β-glucosidase in different combinations

  • Test the effect of adding components of the ROS system (hydroquinone, hydrogen peroxide, iron compounds)

  • Analyze data using response surface methodology to identify optimal combinations

• Time-course degradation studies:

  • Monitor substrate degradation over time with different enzyme combinations

  • Implement sequential addition experiments (e.g., ROS pretreatment followed by enzymes, or Xyn10A pretreatment followed by cellulases)

  • Use time-resolved imaging techniques to visualize changes in substrate structure

• Complementation studies with purified components:

  • Reconstitute the complete G. trabeum degradation system from purified recombinant enzymes

  • Systematically omit individual components to determine their contribution

  • Compare the reconstituted system with crude fungal extracts to identify missing components

• Cross-species synergy analysis:

  • Test G. trabeum Cel5A with cellulase cocktails from industrial strains like Trichoderma reesei

  • Evaluate replacement of components in commercial cocktails with G. trabeum enzymes

  • Study whether the unique properties of G. trabeum Cel5A complement or overlap with other cellulase systems

• Substrate complexity gradients:

  • Design experiments using substrates of increasing complexity

  • Start with defined substrates (filter paper, Avicel) and progress to pretreated and native lignocellulosic materials

  • Analyze how synergistic effects change with substrate recalcitrance

How should researchers interpret changes in product profiles when comparing wild-type and engineered variants of G. trabeum Cel5A?

Interpreting changes in product profiles requires systematic analysis and consideration of multiple factors:

What computational approaches can provide insights into the structure-function relationships of G. trabeum Cel5A?

Computational approaches offer powerful tools for understanding structure-function relationships in G. trabeum Cel5A:

• Homology modeling and structural analysis:

  • Generate a homology model of G. trabeum Cel5A based on crystallized Cel5A enzymes like those from Hypocrea jecorina or Thermotoga maritima

  • Analyze the substrate binding groove architecture and identify key residues for substrate interaction

  • Compare with structures of non-processive endoglucanases to identify unique features that might contribute to processivity

• Molecular dynamics simulations:

  • Simulate enzyme behavior on different cellulosic substrates

  • Analyze protein flexibility and conformational changes during catalysis

  • Identify dynamic networks that connect distal regions to the active site, explaining how mutations far from the active site can enhance activity

• Substrate docking studies:

  • Perform docking simulations with cellooligosaccharides of various lengths

  • Map the complete substrate binding path through the enzyme

  • Identify binding subsites that contribute to processivity

• Quantum mechanics/molecular mechanics (QM/MM) calculations:

  • Model the catalytic mechanism at the electronic level

  • Understand how mutations might affect transition state stabilization

  • Calculate activation energy barriers for key steps in the catalytic cycle

• Bioinformatic analyses:

  • Conduct evolutionary analyses to identify conserved regions across Cel5A enzymes

  • Perform coevolution analysis to identify networks of residues that function together

  • Compare sequences across different GH5 subfamilies to identify determinants of processivity

These computational approaches should be validated through experimental methods such as site-directed mutagenesis and biochemical characterization of the resulting variants.

How can researchers effectively compare the performance of G. trabeum Cel5A with other cellulases on complex lignocellulosic substrates?

Effective comparison of cellulase performance on complex substrates requires standardized methodologies and multifaceted analysis:

• Standardized substrate preparation:

  • Implement consistent pretreatment protocols for lignocellulosic materials

  • Characterize substrate properties (crystallinity, accessibility, chemical composition) before enzymatic treatment

  • Prepare adequate control samples for each batch of experiments

• Multi-parameter performance assessment:

  • Measure sugar release (reducing sugars, specific oligosaccharides, and monomers)

  • Quantify changes in substrate properties (crystallinity index, accessibility measurements)

  • Monitor reaction kinetics by taking time-course measurements

  • Assess performance at different enzyme loadings to generate dose-response curves

• Comparative metrics calculation:

  • Calculate specific activity (μmol product/min/mg enzyme)

  • Determine substrate conversion efficiency (% theoretical maximum)

  • Assess processivity indices on natural substrates

  • Evaluate enzyme stability under reaction conditions

• Advanced analytical techniques:

  • Implement compositional analysis of residual solids

  • Use microscopy techniques to visualize substrate degradation patterns

  • Apply spectroscopic methods to detect chemical modifications

• Statistical analysis approaches:

  • Use multivariate analysis to handle the complexity of performance data

  • Implement principal component analysis to identify key performance differentiators

  • Develop performance indices that combine multiple metrics for simplified comparison

How might insights from G. trabeum Cel5A inform the development of improved enzyme cocktails for biorefinery applications?

The unique characteristics of G. trabeum Cel5A offer several avenues for informing improved enzyme cocktails:

• Integration of processive endoglucanases:

  • Incorporate G. trabeum Cel5A or engineered variants with enhanced processivity into traditional cellulase mixtures

  • Optimize ratios between processive endoglucanases, classical endoglucanases, and cellobiohydrolases for maximal synergy

  • Investigate whether processive endoglucanases can reduce the required loading of expensive cellobiohydrolases

• Mimicking the brown rot degradation system:

  • Develop "hybrid" degradation systems combining enzymatic and oxidative components

  • Implement controlled oxidative pretreatment followed by optimized enzyme cocktails

  • Design enzyme formulations that capitalize on the accessibility patterns created by brown rot attack mechanisms

• Protein engineering strategies:

  • Apply the finding that distal mutations can significantly enhance activity to other industrial cellulases

  • Focus engineering efforts on surface residues and loop regions rather than solely on active site residues

  • Explore the creation of fusion proteins combining beneficial domains from different cellulases

• Process optimization approaches:

  • Develop sequential treatment protocols inspired by the natural progression of brown rot decay

  • Investigate temperature staging based on the thermal properties of different enzymatic components

  • Optimize cocktail formulations for specific pretreatment technologies based on their effects on biomass structure

• Fundamental research directions:

  • Further characterize the structural determinants of processivity in GH5 endoglucanases

  • Investigate the molecular basis of the synergy between enzymatic and oxidative systems

  • Study the evolution of processive endoglucanases to understand how this capability emerged

What are the most promising directions for future research on G. trabeum Cel5A and related enzymes?

Several promising research directions emerge from our current understanding of G. trabeum Cel5A:

• Structural biology investigations:

  • Determine the crystal structure of G. trabeum Cel5A, preferably in complex with substrate

  • Compare with structures of non-processive GH5 endoglucanases to identify processivity determinants

  • Utilize cryo-electron microscopy to visualize enzyme-substrate interactions under near-native conditions

• Advanced protein engineering:

  • Apply directed evolution with improved high-throughput screening methodologies

  • Explore semi-rational design approaches targeting regions identified through structural analysis

  • Investigate domain shuffling with other processive enzymes, including processive GH9 endoglucanases

• Systems biology of brown rot fungi:

  • Conduct transcriptomic and proteomic studies of G. trabeum under various growth conditions

  • Investigate regulatory mechanisms controlling cellulase expression

  • Explore the interplay between enzymatic and non-enzymatic components in natural systems

• Oxidative-enzymatic synergy:

  • Develop detailed mechanistic understanding of how oxidative modifications enhance enzymatic digestibility

  • Design biomimetic systems that replicate the efficiency of brown rot degradation

  • Investigate specific oxidative modifications that enhance Cel5A activity

• Biotechnological applications beyond biofuels:

  • Explore applications in textile processing, paper recycling, and food industries

  • Investigate potential uses in producing specific cello-oligosaccharides for prebiotics or other applications

  • Develop immobilized enzyme systems for continuous processing applications

• Advanced analytical techniques:

  • Develop single-molecule techniques to directly observe processivity

  • Utilize neutron scattering to understand cellulose-enzyme interactions

  • Implement advanced mass spectrometry to characterize oxidatively modified substrates and their interactions with enzymes

These research directions would significantly advance our understanding of G. trabeum Cel5A and contribute to the development of improved biomass conversion technologies.

What experimental approaches can address the current knowledge gaps regarding the structure and mechanism of G. trabeum Cel5A?

Several experimental approaches can address knowledge gaps regarding G. trabeum Cel5A:

• Structural characterization:

  • X-ray crystallography or cryo-electron microscopy to determine the three-dimensional structure

  • Small-angle X-ray scattering (SAXS) to study conformational dynamics in solution

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify regions involved in substrate binding

  • NMR spectroscopy to study protein dynamics and substrate interactions

• Mechanism investigation:

  • Pre-steady-state kinetics to resolve individual steps in the catalytic cycle

  • Single-molecule fluorescence studies to directly observe processivity

  • Isothermal titration calorimetry (ITC) to determine thermodynamic parameters of binding

  • Quartz crystal microbalance with dissipation monitoring (QCM-D) to study real-time binding and processivity

• Comparative biochemistry:

  • Side-by-side comparison with other processive endoglucanases and traditional cellobiohydrolases

  • Creation of chimeric enzymes to identify domains responsible for key properties

  • Systematic mutagenesis of residues potentially involved in processivity

  • Cross-species activity comparisons on various substrates

• Advanced substrate analysis:

  • Solid-state NMR to characterize enzyme-induced changes in cellulose structure

  • Atomic force microscopy to visualize enzyme action on cellulose fibrils

  • Neutron reflectometry to study enzyme binding to model cellulose surfaces

  • Mass spectrometry imaging to map enzyme activity on heterogeneous substrates

• Systems-level studies:

  • Reconstitution of the complete G. trabeum degradation system from purified components

  • In situ studies of wood degradation using microscopy and spectroscopy

  • Transcriptomics and proteomics to understand regulation and expression patterns

  • Metabolomics to track carbon flow during substrate degradation

These experimental approaches, particularly when used in combination, would substantially advance our understanding of G. trabeum Cel5A structure and mechanism, filling current knowledge gaps and providing direction for future applications.