A Calorimetric Assay For Enzymatic Saccharification Of Biomass

Leigh Murphy, Kim Borch, K.C. McFarland, Christina Helena Bohlin, Peter Westh

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

Resumé

A limited selection of assay and screening methodologies for cellulolytic enzymes has been stated as a restriction in biomass research. In this report we test the potential of isothermal calorimetry for this purpose. The primary observable in this technique (the heat flow in Watts), scales with the rate of hydrolysis, and unlike other approaches, it provides a continuous picture of the hydrolytic rate. It was found that the activity of a standard enzyme cocktail against purified cellulose substrates and dilute acid pretreated corn stover (PCS) was readily detected in calorimeters of different types, and that the calorimetric signal scaled with the enzyme activity measured by established analytical techniques. Hence, it was concluded that the heat flow provided a valid measure of the hydrolytic rate also in a complex biomass. The hydrolysis process was consistently found to be exothermic, but the amount of heat released per mole of soluble sugar produced varied for different types of substrates. This variation probably reflects heat contributions from processes which are coupled to the hydrolysis of the glycosidic bond (e.g. dissolution of crystalline substrate or physical transitions in the solid matrix). Calorimetric determinations of absolute reaction rates therefore require initial calibration against another method for each new substrate. However, the main potential of the method lies in real-time measurements of relative changes in hydrolysis rates. This approach may be used both for different starting conditions and the titration of enzyme, substrate, promoters or inhibitors to reacting samples in different stages of the hydrolysis. These experiments are technically straight forward, do not require separate calibrations against other techniques and appear to be useful for studies of the regulation and functional mechanism of cellulases.
OriginalsprogEngelsk
TidsskriftEnzyme and Microbial Technology
Vol/bind46
Udgave nummer2
Sider (fra-til)141-146
ISSN0141-0229
DOI
StatusUdgivet - 2010

Citer dette

Murphy, Leigh ; Borch, Kim ; McFarland, K.C. ; Bohlin, Christina Helena ; Westh, Peter. / A Calorimetric Assay For Enzymatic Saccharification Of Biomass. I: Enzyme and Microbial Technology. 2010 ; Bind 46, Nr. 2. s. 141-146.
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abstract = "A limited selection of assay and screening methodologies for cellulolytic enzymes has been stated as a restriction in biomass research. In this report we test the potential of isothermal calorimetry for this purpose. The primary observable in this technique (the heat flow in Watts), scales with the rate of hydrolysis, and unlike other approaches, it provides a continuous picture of the hydrolytic rate. It was found that the activity of a standard enzyme cocktail against purified cellulose substrates and dilute acid pretreated corn stover (PCS) was readily detected in calorimeters of different types, and that the calorimetric signal scaled with the enzyme activity measured by established analytical techniques. Hence, it was concluded that the heat flow provided a valid measure of the hydrolytic rate also in a complex biomass. The hydrolysis process was consistently found to be exothermic, but the amount of heat released per mole of soluble sugar produced varied for different types of substrates. This variation probably reflects heat contributions from processes which are coupled to the hydrolysis of the glycosidic bond (e.g. dissolution of crystalline substrate or physical transitions in the solid matrix). Calorimetric determinations of absolute reaction rates therefore require initial calibration against another method for each new substrate. However, the main potential of the method lies in real-time measurements of relative changes in hydrolysis rates. This approach may be used both for different starting conditions and the titration of enzyme, substrate, promoters or inhibitors to reacting samples in different stages of the hydrolysis. These experiments are technically straight forward, do not require separate calibrations against other techniques and appear to be useful for studies of the regulation and functional mechanism of cellulases.",
keywords = "Microcalorimetry, Assay development, Hydrolysis of glycosidic bonds, Enzymatic mechanism, Lignocellulose, Cellulase",
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A Calorimetric Assay For Enzymatic Saccharification Of Biomass. / Murphy, Leigh; Borch, Kim; McFarland, K.C.; Bohlin, Christina Helena; Westh, Peter.

I: Enzyme and Microbial Technology, Bind 46, Nr. 2, 2010, s. 141-146.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - A Calorimetric Assay For Enzymatic Saccharification Of Biomass

AU - Murphy, Leigh

AU - Borch, Kim

AU - McFarland, K.C.

AU - Bohlin, Christina Helena

AU - Westh, Peter

PY - 2010

Y1 - 2010

N2 - A limited selection of assay and screening methodologies for cellulolytic enzymes has been stated as a restriction in biomass research. In this report we test the potential of isothermal calorimetry for this purpose. The primary observable in this technique (the heat flow in Watts), scales with the rate of hydrolysis, and unlike other approaches, it provides a continuous picture of the hydrolytic rate. It was found that the activity of a standard enzyme cocktail against purified cellulose substrates and dilute acid pretreated corn stover (PCS) was readily detected in calorimeters of different types, and that the calorimetric signal scaled with the enzyme activity measured by established analytical techniques. Hence, it was concluded that the heat flow provided a valid measure of the hydrolytic rate also in a complex biomass. The hydrolysis process was consistently found to be exothermic, but the amount of heat released per mole of soluble sugar produced varied for different types of substrates. This variation probably reflects heat contributions from processes which are coupled to the hydrolysis of the glycosidic bond (e.g. dissolution of crystalline substrate or physical transitions in the solid matrix). Calorimetric determinations of absolute reaction rates therefore require initial calibration against another method for each new substrate. However, the main potential of the method lies in real-time measurements of relative changes in hydrolysis rates. This approach may be used both for different starting conditions and the titration of enzyme, substrate, promoters or inhibitors to reacting samples in different stages of the hydrolysis. These experiments are technically straight forward, do not require separate calibrations against other techniques and appear to be useful for studies of the regulation and functional mechanism of cellulases.

AB - A limited selection of assay and screening methodologies for cellulolytic enzymes has been stated as a restriction in biomass research. In this report we test the potential of isothermal calorimetry for this purpose. The primary observable in this technique (the heat flow in Watts), scales with the rate of hydrolysis, and unlike other approaches, it provides a continuous picture of the hydrolytic rate. It was found that the activity of a standard enzyme cocktail against purified cellulose substrates and dilute acid pretreated corn stover (PCS) was readily detected in calorimeters of different types, and that the calorimetric signal scaled with the enzyme activity measured by established analytical techniques. Hence, it was concluded that the heat flow provided a valid measure of the hydrolytic rate also in a complex biomass. The hydrolysis process was consistently found to be exothermic, but the amount of heat released per mole of soluble sugar produced varied for different types of substrates. This variation probably reflects heat contributions from processes which are coupled to the hydrolysis of the glycosidic bond (e.g. dissolution of crystalline substrate or physical transitions in the solid matrix). Calorimetric determinations of absolute reaction rates therefore require initial calibration against another method for each new substrate. However, the main potential of the method lies in real-time measurements of relative changes in hydrolysis rates. This approach may be used both for different starting conditions and the titration of enzyme, substrate, promoters or inhibitors to reacting samples in different stages of the hydrolysis. These experiments are technically straight forward, do not require separate calibrations against other techniques and appear to be useful for studies of the regulation and functional mechanism of cellulases.

KW - Microcalorimetry

KW - Assay development

KW - Hydrolysis of glycosidic bonds

KW - Enzymatic mechanism

KW - Lignocellulose

KW - Cellulase

U2 - 10.1016/j.enzmictec.2009.09.009

DO - 10.1016/j.enzmictec.2009.09.009

M3 - Journal article

VL - 46

SP - 141

EP - 146

JO - Enzyme and Microbial Technology

JF - Enzyme and Microbial Technology

SN - 0141-0229

IS - 2

ER -