An enzymatic signal amplification system for calorimetric studies of cellobiohydrolases

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

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

Resumé

The study of cellulolytic enzymes has traditionally been carried out using endpoint measurements by quantitation of reaction products using high-performance liquid chromatography (HPLC) or overall determination of produced reducing ends. To measure catalytic activity, model substrates such as solubilized cellulose derivates, soluble chromogenic, and flourogenic oligomeric substrates are often employed even though they do not reflect the natural insoluble substrate hydrolysis. Thermochemical methods using, for example, isothermal titration calorimetry (ITC) yield data where the primary observable is heat production. This can be converted to the rate of reaction and allows direct and continuous monitoring of the hydrolysis of complex substrates. To overcome the low molar enthalpy of the hydrolysis of the glycosidic bond, which is typically on the order of −2.5 kJ mol−1, an enzymatic signal amplification method has been developed to measure even slow hydrolytically active enzymes such as cellobiohydrolases. This method is explained in detail for the amplification of the heat signal by more than 130 times by using glucose oxidase and catalase. The kinetics of this complex coupled reaction system is thoroughly investigated, and the potential use to generate kinetic models of enzymatic hydrolysis of unmodified cellulosic substrates is demonstrated.

OriginalsprogEngelsk
TidsskriftAnalytical Biochemistry
Vol/bind404
Udgave nummer2
Sider (fra-til)140-148
ISSN0003-2697
DOI
StatusUdgivet - 15 sep. 2010

Citer dette

Murphy, Leigh ; Baumann, Martin Johannes ; Borch, Kim ; Sweeney, Matt ; Westh, Peter. / An enzymatic signal amplification system for calorimetric studies of cellobiohydrolases. I: Analytical Biochemistry. 2010 ; Bind 404, Nr. 2. s. 140-148.
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abstract = "The study of cellulolytic enzymes has traditionally been carried out using endpoint measurements by quantitation of reaction products using high-performance liquid chromatography (HPLC) or overall determination of produced reducing ends. To measure catalytic activity, model substrates such as solubilized cellulose derivates, soluble chromogenic, and flourogenic oligomeric substrates are often employed even though they do not reflect the natural insoluble substrate hydrolysis. Thermochemical methods using, for example, isothermal titration calorimetry (ITC) yield data where the primary observable is heat production. This can be converted to the rate of reaction and allows direct and continuous monitoring of the hydrolysis of complex substrates. To overcome the low molar enthalpy of the hydrolysis of the glycosidic bond, which is typically on the order of −2.5 kJ mol−1, an enzymatic signal amplification method has been developed to measure even slow hydrolytically active enzymes such as cellobiohydrolases. This method is explained in detail for the amplification of the heat signal by more than 130 times by using glucose oxidase and catalase. The kinetics of this complex coupled reaction system is thoroughly investigated, and the potential use to generate kinetic models of enzymatic hydrolysis of unmodified cellulosic substrates is demonstrated.",
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An enzymatic signal amplification system for calorimetric studies of cellobiohydrolases. / Murphy, Leigh; Baumann, Martin Johannes; Borch, Kim; Sweeney, Matt; Westh, Peter.

I: Analytical Biochemistry, Bind 404, Nr. 2, 15.09.2010, s. 140-148.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - An enzymatic signal amplification system for calorimetric studies of cellobiohydrolases

AU - Murphy, Leigh

AU - Baumann, Martin Johannes

AU - Borch, Kim

AU - Sweeney, Matt

AU - Westh, Peter

PY - 2010/9/15

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N2 - The study of cellulolytic enzymes has traditionally been carried out using endpoint measurements by quantitation of reaction products using high-performance liquid chromatography (HPLC) or overall determination of produced reducing ends. To measure catalytic activity, model substrates such as solubilized cellulose derivates, soluble chromogenic, and flourogenic oligomeric substrates are often employed even though they do not reflect the natural insoluble substrate hydrolysis. Thermochemical methods using, for example, isothermal titration calorimetry (ITC) yield data where the primary observable is heat production. This can be converted to the rate of reaction and allows direct and continuous monitoring of the hydrolysis of complex substrates. To overcome the low molar enthalpy of the hydrolysis of the glycosidic bond, which is typically on the order of −2.5 kJ mol−1, an enzymatic signal amplification method has been developed to measure even slow hydrolytically active enzymes such as cellobiohydrolases. This method is explained in detail for the amplification of the heat signal by more than 130 times by using glucose oxidase and catalase. The kinetics of this complex coupled reaction system is thoroughly investigated, and the potential use to generate kinetic models of enzymatic hydrolysis of unmodified cellulosic substrates is demonstrated.

AB - The study of cellulolytic enzymes has traditionally been carried out using endpoint measurements by quantitation of reaction products using high-performance liquid chromatography (HPLC) or overall determination of produced reducing ends. To measure catalytic activity, model substrates such as solubilized cellulose derivates, soluble chromogenic, and flourogenic oligomeric substrates are often employed even though they do not reflect the natural insoluble substrate hydrolysis. Thermochemical methods using, for example, isothermal titration calorimetry (ITC) yield data where the primary observable is heat production. This can be converted to the rate of reaction and allows direct and continuous monitoring of the hydrolysis of complex substrates. To overcome the low molar enthalpy of the hydrolysis of the glycosidic bond, which is typically on the order of −2.5 kJ mol−1, an enzymatic signal amplification method has been developed to measure even slow hydrolytically active enzymes such as cellobiohydrolases. This method is explained in detail for the amplification of the heat signal by more than 130 times by using glucose oxidase and catalase. The kinetics of this complex coupled reaction system is thoroughly investigated, and the potential use to generate kinetic models of enzymatic hydrolysis of unmodified cellulosic substrates is demonstrated.

KW - Isothermal titration calorimetry

KW - Signal amplification

KW - Cellobiohydrolase

KW - Coupled enzyme reactions

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DO - 10.1016/j.ab.2010.04.020

M3 - Journal article

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JO - Analytical Biochemistry

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