title: A graphene screen-printed carbon electrode for real-time measurements of unoccupied active sites in a cellulase.
abstract: Cellulases hydrolyze cellulose to soluble sugars and this process is utilized in sustainable industries based on lignocellulosic feedstock. Better analytical tools will be necessary to understand basic cellulase mechanisms, and hence deliver rational improvements of the industrial process. In this work we describe a new electrochemical approach to the quantification of the populations of enzyme that are respectively free in the aqueous bulk, adsorbed to the insoluble substrate with an unoccupied active site or threaded with the cellulose strand in the active tunnel. Distinction of these three states appears essential to the identification of the rate-limiting step. The method is based on disposable graphene-modified screen-printed carbon electrodes, and we show how the temporal development in the concentrations of the three enzyme forms can be derived from a combination of the electrochemical data and adsorption measurements. The approach was tested for the cellobiohydrolase Cel7A from Hypocrea jecorina acting on microcrystalline cellulose, and it was found that the threaded enzyme form dominates for this system while adsorbed enzyme with an unoccupied active site constitutes less than 5% of the population.
authors.affiliation: Research Unit for Biomaterials, NSM, Roskilde University, Universitetsvej 1, DK-4000 Roskilde, Denmark; Novozymes A/S, Krogshøjvej 36, DK-2880 Bagsværd, Denmark. Electronic address: nicolaj@ruc.dk.
authors.forename: Nicolaj
authors.initials: N
authors.lastname: Cruys-Bagger
authors.affiliation: Department of Chemistry, Faculty of Science, Shinshu University, Matsumoto, Nagano 390-8621, Japan.
authors.forename: Hirosuke
authors.initials: H
authors.lastname: Tatsumi
authors.affiliation: Novozymes A/S, Krogshøjvej 36, DK-2880 Bagsværd, Denmark.
authors.forename: Kim
authors.initials: K
authors.lastname: Borch
authors.affiliation: Research Unit for Biomaterials, NSM, Roskilde University, Universitetsvej 1, DK-4000 Roskilde, Denmark. Electronic address: pwesth@ruc.dk.
authors.forename: Peter
authors.initials: P
authors.lastname: Westh
mesh_terms: D002244:Carbon / Q000737:chemistry*; D020134:Catalytic Domain; D002480:Cellulase / Q000737:chemistry* / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism; D004563:Electrochemistry / Q000295:instrumentation / Q000379:methods*; D004566:Electrodes; D006108:Graphite / Q000737:chemistry*; D035901:Hypocrea / Q000201:enzymology; D008958:Models, Molecular; D011327:Printing
chemical_list: D002244:Carbon; D006108:Graphite; D002482:Cellulose; D002480:Cellulase
keywords: Cellobiohydrolase; Cellulase kinetics; Graphene; Hypocrea jecorina Cel7A; Nitrophenol; Screen-printed carbon electrodes