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

Nicolaj Cruys-Bagger, Ren Guilin, Hirosuke Tatsumi, Martin Baumann, Nikolaj Spodsberg, Heidi Delcomyn Andersen, Lo Gorton, Kim Borch, Peter Westh

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

Resumé

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 +0.5 V (vs. Ag/AgCl). The CDH-biosensors showed high sensitivity (87.7 µA mM−1 cm−2), low detection limit (25 nM), and fast response time (t95% ∼ 3 s) 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 β-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
OriginalsprogEngelsk
TidsskriftBiotechnology and Bioengineering (Print)
Vol/bind109
Udgave nummer12
Sider (fra-til)3199-3204
ISSN0006-3592
DOI
StatusUdgivet - 2012

Citer dette

Cruys-Bagger, Nicolaj ; Guilin, Ren ; Tatsumi, Hirosuke ; Baumann, Martin ; Spodsberg, Nikolaj ; Delcomyn Andersen, Heidi ; Gorton, Lo ; Borch, Kim ; Westh, Peter. / An amperometric enzyme biosensor for real-time measurements of cellobiohydrolase activity on insoluble cellulose. I: Biotechnology and Bioengineering (Print). 2012 ; Bind 109, Nr. 12. s. 3199-3204.
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title = "An amperometric enzyme biosensor for real-time measurements of cellobiohydrolase activity on insoluble cellulose",
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 +0.5 V (vs. Ag/AgCl). The CDH-biosensors showed high sensitivity (87.7 µA mM−1 cm−2), low detection limit (25 nM), and fast response time (t95{\%} ∼ 3 s) 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 β-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",
author = "Nicolaj Cruys-Bagger and Ren Guilin and Hirosuke Tatsumi and Martin Baumann and Nikolaj Spodsberg and {Delcomyn Andersen}, Heidi and Lo Gorton and Kim Borch and Peter Westh",
year = "2012",
doi = "10.1002/bit.24593",
language = "English",
volume = "109",
pages = "3199--3204",
journal = "Biotechnology and Bioengineering (Print)",
issn = "0006-3592",
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Cruys-Bagger, N, Guilin, R, Tatsumi, H, Baumann, M, Spodsberg, N, Delcomyn Andersen, H, Gorton, L, Borch, K & Westh, P 2012, 'An amperometric enzyme biosensor for real-time measurements of cellobiohydrolase activity on insoluble cellulose', Biotechnology and Bioengineering (Print), bind 109, nr. 12, s. 3199-3204. https://doi.org/10.1002/bit.24593

An amperometric enzyme biosensor for real-time measurements of cellobiohydrolase activity on insoluble cellulose. / Cruys-Bagger, Nicolaj; Guilin, Ren; Tatsumi, Hirosuke; Baumann, Martin; Spodsberg, Nikolaj; Delcomyn Andersen, Heidi ; Gorton, Lo; Borch, Kim; Westh, Peter.

I: Biotechnology and Bioengineering (Print), Bind 109, Nr. 12, 2012, s. 3199-3204.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - An amperometric enzyme biosensor for real-time measurements of cellobiohydrolase activity on insoluble cellulose

AU - Cruys-Bagger, Nicolaj

AU - Guilin, Ren

AU - Tatsumi, Hirosuke

AU - Baumann, Martin

AU - Spodsberg, Nikolaj

AU - Delcomyn Andersen, Heidi

AU - Gorton, Lo

AU - Borch, Kim

AU - Westh, Peter

PY - 2012

Y1 - 2012

N2 - 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 +0.5 V (vs. Ag/AgCl). The CDH-biosensors showed high sensitivity (87.7 µA mM−1 cm−2), low detection limit (25 nM), and fast response time (t95% ∼ 3 s) 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 β-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

AB - 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 +0.5 V (vs. Ag/AgCl). The CDH-biosensors showed high sensitivity (87.7 µA mM−1 cm−2), low detection limit (25 nM), and fast response time (t95% ∼ 3 s) 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 β-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

U2 - 10.1002/bit.24593

DO - 10.1002/bit.24593

M3 - Journal article

VL - 109

SP - 3199

EP - 3204

JO - Biotechnology and Bioengineering (Print)

JF - Biotechnology and Bioengineering (Print)

SN - 0006-3592

IS - 12

ER -