title: Insights into enhanced thermostability of a cellulosomal enzyme.
abstract: Improved stability of cellulosomal enzymes is of great significance in order to provide efficient degradation of cellulosic derivatives for production of biofuels. In previous reports, we created a quadruple mutant of the endoglucanase Cel8A from Clostridium thermocellum resulting from a combination of both random error-prone PCR and a bioinformatics-based consensus mutagenesis approach. The quadruple mutant exhibited an increased half-life of activity by 14-fold at 85°C with no apparent loss of catalytic activity compared to the wild-type form. Connection of the wild-type enzyme to its respective cohesin partner conferred increased thermostability, but no increase was observed for the cohesin-complexed mutant enzyme. The mutant and the wild-type enzymes were integrated into divalent chimeric scaffoldins with a family 48 exoglucanase partner, and the cellulose-degradation activities of resultant designer cellulosomes were examined. Despite the heightened thermostability of the mutant as a free enzyme, its substitution for the wild-type endoglucanase within the cellulosome context failed to exhibit an improvement in overall degradation of cellulose.
authors.affiliation: Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel.
authors.forename: Johanna
authors.initials: J
authors.lastname: Stern
authors.affiliation: Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel; Designer Energy, 2, Bergman St., Rehovot, Israel.
authors.forename: Michael
authors.initials: M
authors.lastname: Anbar
authors.affiliation: Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel.
authors.forename: Sarah
authors.initials: S
authors.lastname: Moraïs
authors.affiliation: Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Ramat Aviv 69978, Israel.
authors.forename: Raphael
authors.initials: R
authors.lastname: Lamed
authors.affiliation: Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel. Electronic address: ed.bayer@weizmann.ac.il.
authors.forename: Edward A
authors.initials: EA
authors.lastname: Bayer
mesh_terms: D002266:Carboxymethylcellulose Sodium / Q000378:metabolism; D002480:Cellulase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D044603:Cellulosomes / Q000201:enzymology*; D048013:Acetivibrio thermocellus / Q000166:cytology / Q000201:enzymology; D004795:Enzyme Stability; D009154:Mutation; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013696:Temperature
chemical_list: D011994:Recombinant Proteins; C074775:CelA endoglucanase; D002480:Cellulase; D002266:Carboxymethylcellulose Sodium
keywords: Cellulosome; Cohesin; Dockerin; Endoglucanase; Exoglucanase; Thermostability