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A pyranose dehydrogenase-based biosensor for kinetic analysis of enzymatic hydrolysis of cellulose by cellulases

Paper ID Volume ID Publish Year Pages File Format Full-Text
17207 42651 2014 7 PDF Available
Title
A pyranose dehydrogenase-based biosensor for kinetic analysis of enzymatic hydrolysis of cellulose by cellulases
Abstract

•An electrochemical biosensor based on pyranose dehydrogenase was developed.•The enzyme biosensor is not anomer specific.•The enzyme biosensor showed high sensitivity and stability.•The method can be used for real-time monitoring of cellulases activity on cellulose.

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 +0.25 V (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 (0.39–0.40 s−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.

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Keywords
AmPDH, pyranose dehydrogenase from Agaricus meleagris; BQ, 1,4-benzoquinone; Fc, ferrocene; Fc+, ferricenium ion; Fc+PF6, ferricenium hexafluorophosphate; DCIP, 2,6-dichloroindophenolPyranose dehydrogenase; Biosensor; Cellulase kinetics; Hypocrea jecorina
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A pyranose dehydrogenase-based biosensor for kinetic analysis of enzymatic hydrolysis of cellulose by cellulases
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volumes 58–59, 10 May 2014, Pages 68–74
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us