Application of carbohydrate arrays coupled with mass spectrometry to detect activity of plant-polysaccharide degradative enzymes from the fungus Aspergillus niger

Jolanda M. van Munster,Michel Riese,Baptiste Thomas,Christopher J. Gray,Sabine L. Flitsch,Adrienne L. Davis,David B. Archer

doi:10.1038/srep43117

Jolanda M. van Munster, Michel Riese + Show 5 more

Open Access

https://doi.org/10.1038/srep43117

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Abstract

Renewables-based biotechnology depends on enzymes to degrade plant lignocellulose to simple sugars that are converted to fuels or high-value products. Identification and characterization of such lignocellulose degradative enzymes could be fast-tracked by availability of an enzyme activity measurement method that is fast, label-free, uses minimal resources and allows direct identification of generated products. We developed such a method by applying carbohydrate arrays coupled with MALDI-ToF mass spectrometry to identify reaction products of carbohydrate active enzymes (CAZymes) of the filamentous fungus Aspergillus niger. We describe the production and characterization of plant polysaccharide-derived oligosaccharides and their attachment to hydrophobic self-assembling monolayers on a gold target. We verify effectiveness of this array for detecting exo- and endo-acting glycoside hydrolase activity using commercial enzymes, and demonstrate how this platform is suitable for detection of enzyme activity in relevant biological samples, the culture filtrate of A. niger grown on wheat straw. In conclusion, this versatile method is broadly applicable in screening and characterisation of activity of CAZymes, such as fungal enzymes for plant lignocellulose degradation with relevance to biotechnological applications as biofuel production, the food and animal feed industry.

Highlights

Renewables-based biotechnology depends on enzymes to degrade plant lignocellulose to simple sugars that are converted to fuels or high-value products
We expanded on this work by generating MALDI-ToF MS compatible carbohydrate arrays with plant-derived oligosaccharides, including those with a high degree of polymerisation (DP), and applying them to detect and identify substrates and products of both endo- and exo- acting fungal carbohydrate active enzymes (CAZymes), including a proof of principle application towards detection of activity of lignocellulose-active CAZymes enzymes secreted by A. niger
We show that this system can be used to detect both endo- and exo-acting enzyme activity and we apply the arrays to detect substrate changes caused by activity of CAZymes of A. niger

Summary

Introduction

Renewables-based biotechnology depends on enzymes to degrade plant lignocellulose to simple sugars that are converted to fuels or high-value products. We verify effectiveness of this array for detecting exo- and endo-acting glycoside hydrolase activity using commercial enzymes, and demonstrate how this platform is suitable for detection of enzyme activity in relevant biological samples, the culture filtrate of A. niger grown on wheat straw This versatile method is broadly applicable in screening and characterisation of activity of CAZymes, such as fungal enzymes for plant lignocellulose degradation with relevance to biotechnological applications as biofuel production, the food and animal feed industry. As well as commercially available oligosaccharides, to generate a carbohydrate array on a hydrophobic self-assembling monolayer (SAM) of alkanethiols coating the gold surface of a MALDI-ToF target (Fig. 1) We show that this system can be used to detect both endo- and exo-acting enzyme activity and we apply the arrays to detect substrate changes caused by activity of CAZymes of A. niger

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