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Applied and Environmental Microbiology, September 2002, p. 4292-4300, Vol. 68, No. 9
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.9.4292-4300.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

The Fibronectin Type 3-Like Repeat from the Clostridium thermocellum Cellobiohydrolase CbhA Promotes Hydrolysis of Cellulose by Modifying Its Surface

Irina A. Kataeva,1* Ronald D. Seidel, III,2 Ashit Shah,1 Larry T. West,3 Xin-Liang Li,4 and Lars G. Ljungdahl1

Department of Biochemistry and Molecular Biology and Center for Biological Resources Recovery, The University of Georgia, Athens, Georgia 30602-7229,1 Complex Carbohydrate Research Center, The University of Georgia, Athens, Georgia 30602-4695,2 Department of Crop and Soil Sciences, The University of Georgia, Athens, Georgia 30602-7,3 National Center for Agricultural Utilization Research, USDA-ARS, Peoria, Illinois 616044

Received 11 April 2002/ Accepted 28 June 2002

Fibronectin type 3 homology domains (Fn3) as found in the cellobiohydrolase CbhA of Clostridium thermocellum are common among bacterial extracellular glycohydrolases. The function of these domains is not clear. CbhA is modular and composed of an N-terminal family IV carbohydrate-binding domain (CBDIV), an immunoglobulin-like domain, a family 9 glycosyl hydrolase catalytic domain (Gh9), two Fn3-like domains (Fn31,2), a family III carbohydrate-binding domain (CBDIII), and a dockerin domain. Efficiency of cellulose hydrolysis by truncated forms of CbhA increased in the following order: Gh9 (lowest efficiency), Gh9-Fn31,2 (more efficient), and Gh9-Fn31,2-CBDIII (greatest efficiency). Thermostability of the above constructs decreased in the following order: Gh9 (most stable), Gh9-Fn31,2, and then Gh9-Fn31,2-CBDIII (least stable). Mixing of Orpinomyces endoglucanase CelE with Fn31,2, or Fn31,2-CBDIII increased efficiency of hydrolysis of acid-swollen cellulose (ASC) and filter paper. Scanning electron microscopic studies of filter paper treated with Fn31,2, Fn31,2-CBDIII, or CBDIII showed that the surface of the cellulose fibers had been loosened up and crenellated by Fn31,2 and Fn31,2-CBDIII and to a lesser extent by CBDIII. X-ray diffraction analysis did not reveal changes in the crystallinity of the filter paper. CBDIII bound to ASC and filter paper with capacities of 2.45 and 0.73 µmoles g-1 and relative affinities (Kr) of 1.12 and 2.13 liters g-1, respectively. Fn31,2 bound weakly to both celluloses. Fn31,2-CBD bound to ASC and filter paper with capacities of 3.22 and 0.81 µmoles g-1 and Krs of 1.14 and 1.98 liters g-1, respectively. Fn31,2 and CBDIII contained 2 and 1 mol of calcium per mol, respectively. The results suggest that Fn31,2 aids the hydrolysis of cellulose by modifying its surface. This effect is enhanced by the presence of CBDIII, which increases the concentration of Fn31,2 on the cellulose surface.

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, A210 Life Sciences Building, The University of Georgia, Athens, Georgia 30602-7229. Phone: (706) 542- 1086. Fax: (706) 542-2222. E-mail: kataeva{at}arches.uga.edu.

Applied and Environmental Microbiology, September 2002, p. 4292-4300, Vol. 68, No. 9
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.9.4292-4300.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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