An enzymatic signal amplification system for calorimetric studies of cellobiohydrolases

Murphy, Leigh; Baumann, Martin; Borch, Kim; Sweeney, Matt; Westh, Peter

doi:10.1016/j.ab.2010.04.020

Cited by 28 publications

(29 citation statements)

References 34 publications

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“…The purified enzyme showed a single band in SDS-PAGE (see supplemental data), and the absence of cellobiase activity was confirmed as the lack of detectable activity against the chromogenic substrate analog p-nitrophenyl ␤-D-glucopyranoside. RAC was prepared from Sigmacell 20 (Sigma) using a slight modification of the method of Zhang (13) as described elsewhere (14). The disappearance of the crystal C-4 peak in 13 C cross-polarization/magic angle spinning NMR (14,15) was interpreted as nearly completely amorphous substrate.…”

Section: Methodsmentioning

confidence: 99%

“…RAC was prepared from Sigmacell 20 (Sigma) using a slight modification of the method of Zhang (13) as described elsewhere (14). The disappearance of the crystal C-4 peak in 13 C cross-polarization/magic angle spinning NMR (14,15) was interpreted as nearly completely amorphous substrate. All experiments were conducted in standard buffer with 50 mM sodium acetate and 2 mM CaCl 2 (pH 5.0).…”

Section: Methodsmentioning

confidence: 99%

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Pre-steady-state Kinetics for Hydrolysis of Insoluble Cellulose by Cellobiohydrolase Cel7A

Cruys-Bagger

Elmerdahl

Præstgaard

et al. 2012

Journal of Biological Chemistry

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103

148

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Background:The molecular understanding of factors that limit enzymatic hydrolysis of cellulose remains incomplete. Results: Pre-steady-state analysis of cellulolytic activity provides rate constants for basic steps of the overall reaction. Conclusion: Slow dissociation of inactive enzyme-cellulose complexes governs the hydrolytic rate at pseudo-steady state. Significance: Kinetic constants elucidate molecular mechanisms and structure-function relationships for cellulases.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Pre-steady-state Kinetics for Hydrolysis of Insoluble Cellulose by Cellobiohydrolase Cel7A

Cruys-Bagger

Elmerdahl

Præstgaard

et al. 2012

Journal of Biological Chemistry

Self Cite

103

148

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“…Substrates-Regenerated amorphous cellulose (RAC) was prepared from Sigmacell 20 as described previously (46). Total glucose was quantified (46 -48) using a glucose (Sigma) standard curve at A 490 .…”

Section: Methodsmentioning

confidence: 99%

“…The number-averaged degree of polymerization (DP N ) was calculated from the ratio of reducing ends measured by BCA (see below) and total sugars. The crystallinity index of RAC was determined using solid state 13 C cross-polarization/magic angle spinning nuclear magnetic resonance (NMR) with settings adapted from Matulova et al (49) and described in Murphy et al (46). Cello-oligosaccharides (COS) were prepared according to Zhang and Lynd (50), and purity was determined using high performance anion exchange chromatography with pulsed amperometric detection (46).…”

Section: Methodsmentioning

confidence: 99%

“…A stable base line with no stirring is established, and then the enzyme is injected under maximum stirring and mixed for a total of 60 s. The stirring is then turned off, and the hydrolysis is monitored continuously. All data have been corrected for heat of dilution and calibration factors (46,51,52). Details of the new calorimetric method are given in the supplemental material Fig.…”

Section: Methodsmentioning

confidence: 99%

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Origin of Initial Burst in Activity for Trichoderma reesei endo-Glucanases Hydrolyzing Insoluble Cellulose

Murphy

Cruys-Bagger

Damgaard

et al. 2012

Journal of Biological Chemistry

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The kinetics of cellulose hydrolysis have longbeen described by an initial fast hydrolysis rate, tapering rapidly off, leading to a process that takes days rather than hours to complete. This behavior has been mainly attributed to the action of cellobiohydrolases and often linked to the processive mechanism of this exo-acting group of enzymes. The initial kinetics of endo-glucanases (EGs) is far less investigated, partly due to a limited availability of quantitative assay technologies. We have used isothermal calorimetry to monitor the early time course of the hydrolysis of insoluble cellulose by the three main EGs from Trichoderma reesei (Tr): TrCel7B (formerly EG I), TrCel5A (EG II), and TrCel12A (EG III). These endo-glucanases show a distinctive initial burst with a maximal rate that is about 5-fold higher than the rate after 5 min of hydrolysis. The burst is particularly conspicuous for TrCel7B, which reaches a maximal turnover of about 20 s ؊1 at 30°C and conducts about 1200 catalytic cycles per enzyme molecule in the initial fast phase. For TrCel5A and TrCel12A the extent of the burst is 2-300 cycles per enzyme molecule. The availability of continuous data on EG activity allows an analysis of the mechanisms underlying the initial kinetics, and it is suggested that the slowdown is linked to transient inactivation of enzyme on the cellulose surface. We propose, therefore, that the frequency of structures on the substrate surface that cause transient inactivation determine the extent of the burst phase.

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

Cruys-Bagger¹,

Ren²,

Tatsumi³

et al. 2012

Biotech & Bioengineering

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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 (t(95%) ≈ 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.

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An enzymatic signal amplification system for calorimetric studies of cellobiohydrolases

Cited by 28 publications

References 34 publications

Pre-steady-state Kinetics for Hydrolysis of Insoluble Cellulose by Cellobiohydrolase Cel7A

Pre-steady-state Kinetics for Hydrolysis of Insoluble Cellulose by Cellobiohydrolase Cel7A

Origin of Initial Burst in Activity for Trichoderma reesei endo-Glucanases Hydrolyzing Insoluble Cellulose

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

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