title: A pyranose dehydrogenase-based biosensor for kinetic analysis of enzymatic hydrolysis of cellulose by cellulases.
abstract: A novel electrochemical enzyme biosensor was developed for real-time detection of cellulase activity when acting on their natural insoluble substrate, cellulose. The enzyme biosensor was constructed with pyranose dehydrongease (PDH) from Agaricus meleagris that was immobilized on the surface of a carbon paste electrode, which contained the mediator 2,6-dichlorophenolindophenol (DCIP). An oxidation current of the reduced form of DCIP, DCIPH2, produced by the PDH-catalyzed reaction with either glucose or cellobiose, was recorded under constant-potential amperometry at +0.25V (vs. Ag/AgCl). The PDH-biosensor was shown to be anomer unspecific and it can therefore be used in kinetic studies over broad time-scales of both retaining- and inverting cellulases (in addition to enzyme cocktails). The biosensor was used for real-time measurements of the activity of the inverting cellobiohydrolase Cel6A from Hypocrea jecorina (HjCel6A) on cellulosic substrates with different morphology (bacterial microcrystalline cellulose (BMCC) and Avicel). The steady-state rate of hydrolysis increased towards a saturation plateau with increasing loads of substrate. The experimental results were rationalized using a steady-state rate equation for processive cellulases, and it was found that the turnover for HjCel6A at saturating substrate concentration (i.e. maximal apparent specific activity) was similar (0.39-0.40s(-1)) for the two substrates. Conversely, the substrate load at half-saturation was much lower for BMCC compared to Avicel. Biosensors covered with a polycarbonate membrane showed high operational stability of several weeks with daily use.
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: Research Unit for Biomaterials, NSM, Roskilde University, Universitetsvej 1, DK-4000 Roskilde, Denmark.
authors.forename: Silke Flindt
authors.initials: SF
authors.lastname: Badino
authors.affiliation: Research Unit for Biomaterials, NSM, Roskilde University, Universitetsvej 1, DK-4000 Roskilde, Denmark.
authors.forename: Radina
authors.initials: R
authors.lastname: Tokin
authors.affiliation: Research Unit for Biomaterials, NSM, Roskilde University, Universitetsvej 1, DK-4000 Roskilde, Denmark.
authors.forename: Mark
authors.initials: M
authors.lastname: Gontsarik
authors.affiliation: Research Unit for Biomaterials, NSM, Roskilde University, Universitetsvej 1, DK-4000 Roskilde, Denmark.
authors.forename: Samin
authors.initials: S
authors.lastname: Fathalinejad
authors.affiliation: Novozymes A/S, Krogshøjvej 36, DK-2880 Bagsværd, Denmark.
authors.forename: Kenneth
authors.initials: K
authors.lastname: Jensen
authors.affiliation: Novozymes A/S, Krogshøjvej 36, DK-2880 Bagsværd, Denmark.
authors.forename: Miguel Duarte
authors.initials: MD
authors.lastname: Toscano
authors.affiliation: Research Unit for Biomaterials, NSM, Roskilde University, Universitetsvej 1, DK-4000 Roskilde, Denmark; Novozymes A/S, Krogshøjvej 36, DK-2880 Bagsværd, Denmark.
authors.forename: Trine Holst
authors.initials: TH
authors.lastname: Sørensen
authors.affiliation: Novozymes A/S, Krogshøjvej 36, DK-2880 Bagsværd, Denmark.
authors.forename: Kim
authors.initials: K
authors.lastname: Borch
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: Institute of Molecular and Cell Biology, University of Tartu, Riia 23b - 202, 51010 Tartu, Estonia.
authors.forename: Priit
authors.initials: P
authors.lastname: Väljamäe
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: D015086:2,6-Dichloroindophenol; D000364:Agaricus / Q000201:enzymology; D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D015374:Biosensing Techniques; D002138:Calibration; D002244:Carbon; D002480:Cellulase / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism*; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000378:metabolism; D003199:Computer Systems; D055664:Electrochemical Techniques / Q000295:instrumentation*; D004566:Electrodes; D004867:Equipment Design; D005656:Fungal Proteins / Q000378:metabolism*; D006868:Hydrolysis; D035901:Hypocrea / Q000201:enzymology; D007700:Kinetics; D008567:Membranes, Artificial; D009904:Optical Rotation; D015203:Reproducibility of Results; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D005656:Fungal Proteins; D008567:Membranes, Artificial; D002244:Carbon; D002482:Cellulose; D015086:2,6-Dichloroindophenol; D000429:Alcohol Oxidoreductases; D002480:Cellulase; D043366:Cellulose 1,4-beta-Cellobiosidase; C109691:microcrystalline cellulose
keywords: Biosensor; Cel6A; Cellobiohydrolase; Cellulase kinetics; Hypocrea jecorina; Pyranose dehydrogenase