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Summary: APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 2011, p. 14361442 Vol. 77, No. 4
0099-2240/11/$12.00 doi:10.1128/AEM.01802-10
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Comparison of Family 9 Cellulases from Mesophilic
and Thermophilic Bacteria
Florence Mingardon, John D. Bagert, Cyprien Maisonnier, Devin L. Trudeau, and Frances H. Arnold*
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125
Received 28 July 2010/Accepted 4 December 2010
Cellulases containing a family 9 catalytic domain and a family 3c cellulose binding module (CBM3c) are
important components of bacterial cellulolytic systems. We measured the temperature dependence of the
activities of three homologs: Clostridium cellulolyticum Cel9G, Thermobifida fusca Cel9A, and C. thermocellum
Cel9I. To directly compare their catalytic activities, we constructed six new versions of the enzymes in which
the three GH9-CBM3c domains were fused to a dockerin both with and without a T. fusca fibronectin type 3
homology module (Fn3). We studied the activities of these enzymes on crystalline cellulose alone and in
complex with a miniscaffoldin containing a cohesin and a CBM3a. The presence of Fn3 had no measurable
effect on thermostability or cellulase activity. The GH9-CBM3c domains of Cel9A and Cel9I, however, were
more active than the wild type when fused to a dockerin complexed to scaffoldin. The three cellulases in
complex have similar activities on crystalline cellulose up to 60°C, but C. thermocellum Cel9I, the most
thermostable of the three, remains highly active up to 80°C, where its activity is 1.9 times higher than at 60°C.
We also compared the temperature-dependent activities of different versions of Cel9I (wild type or in complex
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