A practical approach to steady-state kinetic analysis of cellulases acting on their natural insoluble substrate

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

Resumé

Measurement of steady-state rates (vSS) is straightforward in standard enzymology with soluble substrate, and it has been instrumental for comparative biochemical analyses within this area. For insoluble substrate, however, experimental values of vss remain controversial, and this has strongly limited the amount and quality of comparative analyses for cellulases and other enzymes that act on the surface of an insoluble substrate. In the current work, we have measured progress curves over a wide range of conditions for two cellulases, TrCel6A and TrCel7A from Trichoderma reesei, acting on their natural, insoluble substrate, cellulose. Based on this, we consider practical compromises for the determination of experimental vSS values, and propose a basic protocol that provides representative reaction rates and is experimentally simple so that larger groups of enzymes and conditions can be readily assayed with standard laboratory equipment. We surmise that the suggested experimental approach can be useful in comparative biochemical studies of cellulases; an area that remains poorly developed.
OriginalsprogEngelsk
Artikelnummer113411
TidsskriftAnalytical Biochemistry
Vol/bind2019
Udgave nummer586
ISSN0003-2697
DOI
StatusUdgivet - 2019

Citer dette

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title = "A practical approach to steady-state kinetic analysis of cellulases acting on their natural insoluble substrate",
abstract = "Measurement of steady-state rates (vSS) is straightforward in standard enzymology with soluble substrate, and it has been instrumental for comparative biochemical analyses within this area. For insoluble substrate, however, experimental values of vss remain controversial, and this has strongly limited the amount and quality of comparative analyses for cellulases and other enzymes that act on the surface of an insoluble substrate. In the current work, we have measured progress curves over a wide range of conditions for two cellulases, TrCel6A and TrCel7A from Trichoderma reesei, acting on their natural, insoluble substrate, cellulose. Based on this, we consider practical compromises for the determination of experimental vSS values, and propose a basic protocol that provides representative reaction rates and is experimentally simple so that larger groups of enzymes and conditions can be readily assayed with standard laboratory equipment. We surmise that the suggested experimental approach can be useful in comparative biochemical studies of cellulases; an area that remains poorly developed.",
author = "Jeppe Kari and Christensen, {Stefan Jarl} and {Wienecke Andersen}, Morten and {Sell{\'e}s Baiget}, Selene and Kim Borch and Peter Westh",
year = "2019",
doi = "10.1016/j.ab.2019.113411",
language = "English",
volume = "2019",
journal = "Analytical Biochemistry",
issn = "0003-2697",
publisher = "Elsevier Inc.",
number = "586",

}

A practical approach to steady-state kinetic analysis of cellulases acting on their natural insoluble substrate. / Kari, Jeppe; Christensen, Stefan Jarl; Wienecke Andersen, Morten; Sellés Baiget, Selene; Borch, Kim; Westh, Peter.

I: Analytical Biochemistry, Bind 2019, Nr. 586, 113411, 2019.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - A practical approach to steady-state kinetic analysis of cellulases acting on their natural insoluble substrate

AU - Kari, Jeppe

AU - Christensen, Stefan Jarl

AU - Wienecke Andersen, Morten

AU - Sellés Baiget, Selene

AU - Borch, Kim

AU - Westh, Peter

PY - 2019

Y1 - 2019

N2 - Measurement of steady-state rates (vSS) is straightforward in standard enzymology with soluble substrate, and it has been instrumental for comparative biochemical analyses within this area. For insoluble substrate, however, experimental values of vss remain controversial, and this has strongly limited the amount and quality of comparative analyses for cellulases and other enzymes that act on the surface of an insoluble substrate. In the current work, we have measured progress curves over a wide range of conditions for two cellulases, TrCel6A and TrCel7A from Trichoderma reesei, acting on their natural, insoluble substrate, cellulose. Based on this, we consider practical compromises for the determination of experimental vSS values, and propose a basic protocol that provides representative reaction rates and is experimentally simple so that larger groups of enzymes and conditions can be readily assayed with standard laboratory equipment. We surmise that the suggested experimental approach can be useful in comparative biochemical studies of cellulases; an area that remains poorly developed.

AB - Measurement of steady-state rates (vSS) is straightforward in standard enzymology with soluble substrate, and it has been instrumental for comparative biochemical analyses within this area. For insoluble substrate, however, experimental values of vss remain controversial, and this has strongly limited the amount and quality of comparative analyses for cellulases and other enzymes that act on the surface of an insoluble substrate. In the current work, we have measured progress curves over a wide range of conditions for two cellulases, TrCel6A and TrCel7A from Trichoderma reesei, acting on their natural, insoluble substrate, cellulose. Based on this, we consider practical compromises for the determination of experimental vSS values, and propose a basic protocol that provides representative reaction rates and is experimentally simple so that larger groups of enzymes and conditions can be readily assayed with standard laboratory equipment. We surmise that the suggested experimental approach can be useful in comparative biochemical studies of cellulases; an area that remains poorly developed.

U2 - 10.1016/j.ab.2019.113411

DO - 10.1016/j.ab.2019.113411

M3 - Journal article

VL - 2019

JO - Analytical Biochemistry

JF - Analytical Biochemistry

SN - 0003-2697

IS - 586

M1 - 113411

ER -