Activity-based protein profiling of secreted cellulolytic enzyme activity dynamics in Trichoderma reesei QM6a, NG14, and RUT-C30

Anderson, Lindsey; Culley, David E.; Hofstad, Beth A.; Chauvigné-Hines, Lacie M.; Zink, Erika; Smith, Richard D.; Callister, Stephen; Magnuson, Jon; Wright, Aaron

doi:10.1039/c3mb70333a

Cited by 12 publications

(12 citation statements)

References 44 publications

(67 reference statements)

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“…[33] This low pH requirement might be limiting for this system for some applications as fungal cellulases have pH optima in the range of 4 to 6.5. [34] However, the HyReS system pH range (< pH 5) matches optimal conditions for many cellulolytic enzyme formulations (e.g., Tr and A. niger cocktails). [35,36] Developing systems for real-time imaging of cellulose degradation is an important step towards improved enzyme formulations for biofuel development.…”

Section: Resultsmentioning

confidence: 99%

Redox‐Initiated Hydrogel System for Detection and Real‐Time Imaging of Cellulolytic Enzyme Activity

et al. 2014

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Understanding the process of biomass degradation by cellulolytic enzymes is of urgent importance for biofuel and chemical production. Optimizing pretreatment conditions and improving enzyme formulations both require assays to quantify saccharification products on solid substrates. Typically, such assays are performed using freely diffusing fluorophores or dyes that measure reducing polysaccharide chain ends. These methods have thus far not allowed spatial localization of hydrolysis activity to specific substrate locations with identifiable morphological features. Here we describe a hydrogel reagent signaling (HyReS) system that amplifies saccharification products and initiates crosslinking of a hydrogel that localizes to locations of cellulose hydrolysis, allowing for imaging of the degradation process in real time. Optical detection of the gel in a rapid parallel format on synthetic and natural pretreated solid substrates was used to quantify activity of T. emersonii and T. reesei enzyme cocktails. When combined with total internal reflection fluorescence microscopy and AFM imaging, the reagent system provided a means to visualize enzyme activity in real-time with high spatial resolution (<2 μm). These results demonstrate the versatility of the HyReS system in detecting cellulolytic enzyme activity and suggest new opportunities in real-time chemical imaging of biomass depolymerization.

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Section: Resultsmentioning

confidence: 99%

Redox‐Initiated Hydrogel System for Detection and Real‐Time Imaging of Cellulolytic Enzyme Activity

et al. 2014

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“…These studies successfully used the probes to enrich carrier proteins and allowed for the visualization and proteomic identification of fatty acid ACPs in E. coli, B. subtilis, S. oneidensis, and B. brevis lysates [21][22][23]. to facilitate biorefinery and biotechnology applications of glycoside hydrolases used the same probes to characterize the functional response of enzymes to mild and significant perturbations in temperature, pH, and culture duration in the fungus Trichoderma reesei [25]. The suite of probes for glycoside hydrolases provides opportunities for optimizing biofuel processing conditions, and identifying enzymes for incorporation into enzyme "cocktails" for potentially improving biofuel production.…”

Section: Fatty Acid Synthasesmentioning

confidence: 99%

Activity-based protein profiling of microbes

Sadler

Wright

2015

Current Opinion in Chemical Biology

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Activity-based protein profiling (ABPP) in conjunction with multimodal characterization techniques has yielded impactful findings in microbiology, particularly in pathogen, bioenergy, drug discovery, and environmental research. Using small molecule chemical probes that react irreversibly with specific proteins or protein families in complex systems has provided insights in enzyme functions in central metabolic pathways, drug-protein interactions, and regulatory protein redox, for systems ranging from photoautotrophic cyanobacteria to mycobacteria, and combining live cell or cell extract ABPP with proteomics, molecular biology, modeling, and other techniques has greatly expanded our understanding of these systems. New opportunities for application of ABPP to microbial systems can enhance protein annotation, characterize protein activities in myriad environments, and reveal signal transduction and regulatory mechanisms in microbial systems.

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“…Therefore, it may not provide sufficient resolution of enzyme mixtures because the expression level of each enzyme cannot be correlated to its own activity. Recently, the Activity-Based Protein Profiling (ABPP) approach has been adopted to profile the biomass degrading enzymes in the fungal secretome [8–10]. It utilized the activity-based probes (ABPs) to covalently label and affinity-purify the GHs from fungal secretome for proteomics screening.…”

Section: Introductionmentioning

confidence: 99%

Finding Biomass Degrading Enzymes Through an Activity-Correlated Quantitative Proteomics Platform (ACPP)

Delafield

Wang

You

et al. 2017

J. Am. Soc. Mass Spectrom.

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The microbial secretome, known as a pool of biomass (i.e., plant-based materials) degrading enzymes, can be utilized to discover industrial enzyme candidates for biofuel production. Proteomics approaches have been applied to discover novel enzyme candidates through comparing protein expression profiles with the whole secretome enzymatic activity under different growth conditions. However, the activity measurement of each enzyme candidate is needed for confident “active” enzyme assignments, and it remains to be elucidated. To address this challenge, we have developed an Activity-Correlated Quantitative Proteomics Platform (ACPP) that systematically correlate protein-level enzymatic activity patterns and protein elution profiles using a label-free quantitative proteomics approach. The ACPP optimized a high performance anion exchange separation for efficiently fractionating complex protein samples while preserving enzymatic activities. The detected enzymatic activity patterns in sequential fractions using microplate-based assays were cross-correlated with protein elution profiles using a customized pattern-matching algorithm with a correlation R score. The ACPP has been successfully applied to the identification of two types of “active” biomass-degrading enzymes (i.e., starch hydrolysis enzymes and cellulose hydrolysis enzymes) from Aspergillus niger secretome in a multiplexed fashion. By determining protein elution profiles of 156 proteins in A. niger secretome, we confidently identified the 1, 4-α-glucosidase as the major “active” starch hydrolysis enzyme (R=0.96) and the endoglucanase as the major “active” cellulose hydrolysis enzyme (R=0.97). The results demonstrated that the ACPP facilitated the discovery of bioactive enzymes from complex protein samples in a high-throughput, multiplexing, and untargeted fashion.

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Activity-based protein profiling of secreted cellulolytic enzyme activity dynamics in Trichoderma reesei QM6a, NG14, and RUT-C30

Cited by 12 publications

References 44 publications

Redox‐Initiated Hydrogel System for Detection and Real‐Time Imaging of Cellulolytic Enzyme Activity

Redox‐Initiated Hydrogel System for Detection and Real‐Time Imaging of Cellulolytic Enzyme Activity

Activity-based protein profiling of microbes

Finding Biomass Degrading Enzymes Through an Activity-Correlated Quantitative Proteomics Platform (ACPP)

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