title: Application of carbohydrate arrays coupled with mass spectrometry to detect activity of plant-polysaccharide degradative enzymes from the fungus Aspergillus niger.
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.
authors.affiliation: Fungal Biology and Genetics, School of Life Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK.
authors.forename: Jolanda M
authors.initials: JM
authors.lastname: van Munster
authors.affiliation: Chemical Biology, Manchester Institute for Biotechnology, University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom.
authors.forename: Baptiste
authors.initials: B
authors.lastname: Thomas
authors.affiliation: Chemical Biology, Manchester Institute for Biotechnology, University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom.
authors.forename: Michel
authors.initials: M
authors.lastname: Riese
authors.affiliation: School of Chemistry, University of Nottingham, University Park, Nottingham NG7 2RD, UK.
authors.forename: Adrienne L
authors.initials: AL
authors.lastname: Davis
authors.affiliation: Chemical Biology, Manchester Institute for Biotechnology, University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Gray
authors.affiliation: Fungal Biology and Genetics, School of Life Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK.
authors.forename: David B
authors.initials: DB
authors.lastname: Archer
authors.affiliation: Chemical Biology, Manchester Institute for Biotechnology, University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom.
authors.forename: Sabine L
authors.initials: SL
authors.lastname: Flitsch
mesh_terms: D001234:Aspergillus niger / Q000201:enzymology*; D001709:Biotechnology / Q000379:methods; D005656:Fungal Proteins / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism*; D008031:Lignin / Q000032:analysis / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D005656:Fungal Proteins; C036909:lignocellulose; D008031:Lignin; D006026:Glycoside Hydrolases