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Applied and Environmental Microbiology, April 2000, p. 1444-1452, Vol. 66, No. 4
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Imaging the Enzymatic Digestion of Bacterial Cellulose Ribbons Reveals the Endo Character of the Cellobiohydrolase Cel6A from Humicola insolens and Its Mode of Synergy with Cellobiohydrolase Cel7A

Claire Boisset,1,* Carole Fraschini,1 Martin Schülein,2 Bernard Henrissat,3 and Henri Chanzy1

Centre de Recherches sur les Macromolécules Végétales (CNRS), Joseph Fourier University of Grenoble, F-38041 Grenoble Cedex,1 and Architecture et Fonction des Macromolécules Biologiques, CNRS-IFR1, F-13402 Marseille Cedex 20,3 France, and Novo Nordisk, DK-2880 Bagsvaerd, Denmark2

Received 3 November 1999/Accepted 18 January 2000

Dispersed cellulose ribbons from bacterial cellulose were subjected to digestion with cloned Cel7A (cellobiohydrolase [CBH] I) and Cel6A (CBH II) from Humicola insolens either alone or in a mixture and in the presence of an excess of beta -glucosidase. Both Cel7A and Cel6A were effective in partially converting the ribbons into soluble sugars, Cel7A being more active than Cel6A. In combination, these enzymes showed substantial synergy culminating with a molar ratio of approximately two-thirds Cel6A and one-third Cel7A. Ultrastructural transmission electron microscopy (TEM) observations indicated that Cel7A induced a thinning of the cellulose ribbons, whereas Cel6A cut the ribbons into shorter elements, indicating an endo type of action. These observations, together with the examination of the digestion kinetics, indicate that Cel6A can be classified as an endo-processive enzyme, whereas Cel7A is essentially a processive enzyme. Thus, the synergy resulting from the mixing of Cel6A and Cel7A can be explained by the partial endo character of Cel6A. A preparation of bacterial cellulose ribbons appears to be an appropriate substrate, superior to Valonia or bacterial cellulose microcrystals, to visualize directly by TEM the endo-processivity of an enzyme such as Cel6A.

* Corresponding author. Mailing address: Centre de Recherches sur les Macromolécules Végétales (CNRS), B. P. 53, F-38041 Grenoble Cedex 9, France. Phone: (33) 476 03 76 03. Fax: (33) 476 54 72 03. E-mail: Claire.Boisset{at}cermav.cnrs.fr.

Applied and Environmental Microbiology, April 2000, p. 1444-1452, Vol. 66, No. 4
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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