title: Application of time-of-flight-secondary ion mass spectrometry for the detection of enzyme activity on solid wood substrates.
abstract: Time-of-flight-secondary ion mass spectrometry (TOF-SIMS) is a surface analysis technique that is herein demonstrated to be a viable tool for the detection of enzyme activity on solid substrates. Proof-of-principle experiments are presented that utilize commercial cellulase and laccase enzymes, which are known to modify major polymeric components of wood (i.e., cellulose and lignin, respectively). Enzyme activity is assessed through principle component analysis (PCA) as well as through peak ratios intended to measure selective enzymatic wood degradation. Spectral reproducibility of the complex wood substrates is found to be within 5% relative standard deviation (RSD), allowing for relative quantification of changes in wood composition. Procedures are also presented to identify and avoid the influence of mass interferences from protein adsorption by the enzyme solutions. The activity of a cellulase cocktail is clearly evident through the TOF-SIMS spectra and is supported by high-pressure liquid chromatography (HPLC) measurements of sugar release and by complementary X-ray photoelectron spectroscopy (XPS) measurements of the wood surfaces. Laccase activity, which is mediated through small organic molecules, can be detected in the TOF-SIMS spectra through a decrease in G and S lignin peaks. This work has positive implications for the development of qualitative, high-throughput screening assays for enzyme activity on industrially relevant, lignocellulosic substrates.
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Robyn E
authors.initials: RE
authors.lastname: Goacher
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Edwards
authors.forename: Alexander F
authors.initials: AF
authors.lastname: Yakunin
authors.forename: Charles A
authors.initials: CA
authors.lastname: Mims
authors.forename: Emma R
authors.initials: ER
authors.lastname: Master
mesh_terms: D002480:Cellulase / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004798:Enzymes / Q000032:analysis* / Q000378:metabolism; D042845:Laccase / Q000378:metabolism; D056951:Photoelectron Spectroscopy; D025341:Principal Component Analysis; D018629:Spectrometry, Mass, Secondary Ion; D013379:Substrate Specificity; D014934:Wood / Q000737:chemistry / Q000378:metabolism*
chemical_list: D004798:Enzymes; D042845:Laccase; D002480:Cellulase