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Activity-based protein profiling reveals dynamic substrate-specific cellulase secretion by saprotrophic
Nicholas G S McGregor1, Casper de Boer2, Mikhaaeel Santos1
1York Structural Biology Laboratory, Department of Chemistry, The University of York, Heslington, YO10 5DD, York, UK.
Biotechnology for Biofuels and Bioproducts
|April 14, 2022
Summary
This study introduces a new activity-based probe (ABP-Cel) for profiling fungal endo-β-glucanases. It reveals unique enzyme fingerprints in fungi grown on lignocellulosic biomass, aiding the biomass saccharification industry.
Area of Science:
- Biotechnology
- Biochemistry
- Mycology
Background:
- Fungal secretomes contain diverse proteins for lignocellulosic biomass saccharification.
- Understanding fungal responses to different substrates is crucial for the biomass industry.
- Cellulases, including endo-β-glucanases, are key components of fungal enzyme systems.
Purpose of the Study:
- To profile endo-β-glucanase activity in fungal secretomes using a novel activity-based probe (ABP-Cel).
- To investigate fungal enzyme secretion dynamics across different species and substrates.
- To establish a sensitive method for detecting fungal enzyme activity in early growth stages.
Main Methods:
- Utilized ABP-Cel, an activity-based probe for endo-β-glucanases.
- Combined ABP-Cel with probes for β-xylanases and β-D-glucosidases for multiplexed profiling.
- Analyzed 240 fungal secretomes from ten basidiomycete fungi grown on maltose, wheat straw, or aspen pulp.
- Performed chemical proteomic analysis and characterized recombinant enzymes.
Main Results:
- ABP-Cel successfully enabled endo-β-glucanase profiling in diverse fungal secretomes.
- Multiplexed activity-based protein profiling revealed unique enzyme fingerprints for each species-substrate combination.
- Significant cellulase arsenals were identified, with ABP-Cel showing broad selectivity for endo-β-glucanase activity across different enzyme families (GH5, GH10, GH12).
Conclusions:
- ABP-Cel provides sensitive, direct evidence of fungal enzyme secretion.
- The method requires minimal sample preparation and small sample volumes.
- This approach is valuable for studying fungal enzyme secretion during early growth on lignocellulosic substrates.
