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An amperometric enzyme biosensor for real-time measurements of cellobiohydrolase activity on insoluble cellulose

Cruys-Bagger, Nicolaj; Ren, Guilin; Tatsumi, Hirosuke; Baumann, Martin J.; Spodsberg, Nikolaj; Andersen, Heidi Delcomyn; Gorton, Lo LU ; Borch, Kim and Westh, Peter (2012) In Biotechnology and Bioengineering 109(12). p.3199-3204
Abstract
An amperometric enzyme biosensor for continuous detection of cellobiose has been implemented as an enzyme assay for cellulases. We show that the initial kinetics for cellobiohydrolase I, Cel7A from Trichoderma reesei, acting on different types of cellulose substrates, semi-crystalline and amorphous, can be monitored directly and in real-time by an enzyme-modified electrode based on cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium (Pc). PcCDH was cross-linked and immobilized on the surface of a carbon paste electrode which contained a mediator, benzoquinone. An oxidation current of the reduced mediator, hydroquinone, produced by the CDH-catalyzed reaction with cellobiose, was recorded under constant-potential amperometry at... (More)
An amperometric enzyme biosensor for continuous detection of cellobiose has been implemented as an enzyme assay for cellulases. We show that the initial kinetics for cellobiohydrolase I, Cel7A from Trichoderma reesei, acting on different types of cellulose substrates, semi-crystalline and amorphous, can be monitored directly and in real-time by an enzyme-modified electrode based on cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium (Pc). PcCDH was cross-linked and immobilized on the surface of a carbon paste electrode which contained a mediator, benzoquinone. An oxidation current of the reduced mediator, hydroquinone, produced by the CDH-catalyzed reaction with cellobiose, was recorded under constant-potential amperometry at +0.5V (vs. Ag/AgCl). The CDH-biosensors showed high sensitivity (87.7 mu AmM-1cm-2), low detection limit (25nM), and fast response time (t95%similar to 3s) and this provided experimental access to the transient kinetics of cellobiohydrolases acting on insoluble cellulose. The response from the CDH-biosensor during enzymatic hydrolysis was corrected for the specificity of PcCDH for the beta-anomer of cello-oligosaccharides and the approach were validated against HPLC. It is suggested that quantitative, real-time data on pure insoluble cellulose substrates will be useful in attempts to probe the molecular mechanism underlying enzymatic hydrolysis of cellulose. Biotechnol. Bioeng. 2012; 109: 31993204. (C) 2012 Wiley Periodicals, Inc. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
cellulose hydrolysis, cellulase kinetics, enzyme biosensor, cellobiose, dehydrogenase
in
Biotechnology and Bioengineering
volume
109
issue
12
pages
3199 - 3204
publisher
John Wiley & Sons
external identifiers
  • wos:000310465300032
  • scopus:84868122545
ISSN
1097-0290
DOI
10.1002/bit.24593
language
English
LU publication?
yes
id
abc0c5e9-9a3d-49f4-a9fe-c1e395fa3be4 (old id 3283362)
date added to LUP
2012-12-20 12:14:41
date last changed
2017-09-10 03:00:18
@article{abc0c5e9-9a3d-49f4-a9fe-c1e395fa3be4,
  abstract     = {An amperometric enzyme biosensor for continuous detection of cellobiose has been implemented as an enzyme assay for cellulases. We show that the initial kinetics for cellobiohydrolase I, Cel7A from Trichoderma reesei, acting on different types of cellulose substrates, semi-crystalline and amorphous, can be monitored directly and in real-time by an enzyme-modified electrode based on cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium (Pc). PcCDH was cross-linked and immobilized on the surface of a carbon paste electrode which contained a mediator, benzoquinone. An oxidation current of the reduced mediator, hydroquinone, produced by the CDH-catalyzed reaction with cellobiose, was recorded under constant-potential amperometry at +0.5V (vs. Ag/AgCl). The CDH-biosensors showed high sensitivity (87.7 mu AmM-1cm-2), low detection limit (25nM), and fast response time (t95%similar to 3s) and this provided experimental access to the transient kinetics of cellobiohydrolases acting on insoluble cellulose. The response from the CDH-biosensor during enzymatic hydrolysis was corrected for the specificity of PcCDH for the beta-anomer of cello-oligosaccharides and the approach were validated against HPLC. It is suggested that quantitative, real-time data on pure insoluble cellulose substrates will be useful in attempts to probe the molecular mechanism underlying enzymatic hydrolysis of cellulose. Biotechnol. Bioeng. 2012; 109: 31993204. (C) 2012 Wiley Periodicals, Inc.},
  author       = {Cruys-Bagger, Nicolaj and Ren, Guilin and Tatsumi, Hirosuke and Baumann, Martin J. and Spodsberg, Nikolaj and Andersen, Heidi Delcomyn and Gorton, Lo and Borch, Kim and Westh, Peter},
  issn         = {1097-0290},
  keyword      = {cellulose hydrolysis,cellulase kinetics,enzyme biosensor,cellobiose,dehydrogenase},
  language     = {eng},
  number       = {12},
  pages        = {3199--3204},
  publisher    = {John Wiley & Sons},
  series       = {Biotechnology and Bioengineering},
  title        = {An amperometric enzyme biosensor for real-time measurements of cellobiohydrolase activity on insoluble cellulose},
  url          = {http://dx.doi.org/10.1002/bit.24593},
  volume       = {109},
  year         = {2012},
}