Reaction conditions for the reducing-end-specific derivatization of cellulose substrates with the fluorogenic compound, anthranilic acid, have been established. Hydrolysis of fluorescence-labelled celluloses by cellobiohydrolase Cel7A from Trichoderma reesei was consistent with the active-site titration kinetics (burst kinetics), which allowed the quantification of the processivity of the enzyme. The processivity values of 88±10, 42±10 and 34±2.0 cellobiose units were found for Cel7A acting on labelled bacterial cellulose, bacterial microcrystalline cellulose and endoglucanase-pretreated bacterial cellulose respectively. The anthranilic acid derivatization also provides an alternative means for estimating the average degree of polymerization of cellulose and, furthermore, allows the quantitative monitoring of the production of reducing end groups on solid cellulose on hydrolysis by cellulases. Hydrolysis of bacterial cellulose by cellulases from T. reesei revealed that, by contrast with endoglucanase Cel5A, neither cellobiohydrolases Cel7A nor Cel6A produced detectable amounts of new reducing end groups on residual cellulose.
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Research Article|
January 07 2005
Processive action of cellobiohydrolase Cel7A from Trichoderma reesei is revealed as ‘burst’ kinetics on fluorescent polymeric model substrates
Kalle KIPPER;
Kalle KIPPER
*Institute of Molecular and Cell Biology, University of Tartu, Riia 23, Tartu 51010, Estonia
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Priit VÄLJAMÄE;
Priit VÄLJAMÄE
1
*Institute of Molecular and Cell Biology, University of Tartu, Riia 23, Tartu 51010, Estonia
1To whom correspondence should be addressed (email priit.valjamae@ut.ee).
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Gunnar JOHANSSON
Gunnar JOHANSSON
†Department of Biochemistry, University of Uppsala, P.O. Box 576, S-75 123 Uppsala, Sweden
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Publisher: Portland Press Ltd
Received:
July 05 2004
Revision Received:
September 13 2004
Accepted:
September 14 2004
Accepted Manuscript online:
September 14 2004
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2005
Biochem J (2005) 385 (2): 527–535.
Article history
Received:
July 05 2004
Revision Received:
September 13 2004
Accepted:
September 14 2004
Accepted Manuscript online:
September 14 2004
Citation
Kalle KIPPER, Priit VÄLJAMÄE, Gunnar JOHANSSON; Processive action of cellobiohydrolase Cel7A from Trichoderma reesei is revealed as ‘burst’ kinetics on fluorescent polymeric model substrates. Biochem J 15 January 2005; 385 (2): 527–535. doi: https://doi.org/10.1042/BJ20041144
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