title: Comparison of family 9 cellulases from mesophilic and thermophilic bacteria.
abstract: 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 with a miniscaffoldin) and found that the thermostable CBM is necessary for activity on crystalline cellulose at high temperatures. These results illustrate the significant benefits of working with thermostable enzymes at high temperatures, as well as the importance of retaining the stability of all modules involved in cellulose degradation.
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
authors.forename: Florence
authors.initials: F
authors.lastname: Mingardon
authors.forename: John D
authors.initials: JD
authors.lastname: Bagert
authors.forename: Cyprien
authors.initials: C
authors.lastname: Maisonnier
authors.forename: Devin L
authors.initials: DL
authors.lastname: Trudeau
authors.forename: Frances H
authors.initials: FH
authors.lastname: Arnold
mesh_terms: D000192:Actinomycetales / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002480:Cellulase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism; D046973:Ruminiclostridium cellulolyticum / Q000201:enzymology*; D048013:Acetivibrio thermocellus / Q000201:enzymology*; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000378:metabolism; D006358:Hot Temperature; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D002482:Cellulose; D006026:Glycoside Hydrolases; C450429:Cel9M protein, Clostridium cellulolyticum; D002480:Cellulase