title: Enhancement of cellulosome-mediated deconstruction of cellulose by improving enzyme thermostability.
abstract: BACKGROUND
  The concerted action of three complementary cellulases from Clostridium thermocellum, engineered to be stable at elevated temperatures, was examined on a cellulosic substrate and compared to that of the wild-type enzymes. Exoglucanase Cel48S and endoglucanase Cel8A, both key elements of the natural cellulosome from this bacterium, were engineered previously for increased thermostability, either by SCHEMA, a structure-guided, site-directed protein recombination method, or by consensus-guided mutagenesis combined with random mutagenesis using error-prone PCR, respectively. A thermostable β-glucosidase BglA mutant was also selected from a library generated by error-prone PCR that will assist the two cellulases in their methodic deconstruction of crystalline cellulose. The effects of a thermostable scaffoldin versus those of a largely mesophilic scaffoldin were also examined. By improving the stability of the enzyme subunits and the structural component, we aimed to improve cellulosome-mediated deconstruction of cellulosic substrates.
  
  
  RESULTS
  The results demonstrate that the combination of thermostable enzymes as free enzymes and a thermostable scaffoldin was more active on the cellulosic substrate than the wild-type enzymes. Significantly, "thermostable" designer cellulosomes exhibited a 1.7-fold enhancement in cellulose degradation compared to the action of conventional designer cellulosomes that contain the respective wild-type enzymes. For designer cellulosome formats, the use of the thermostabilized scaffoldin proved critical for enhanced enzymatic performance under conditions of high temperatures.
  
  
  CONCLUSIONS
  Simple improvement in the activity of a given enzyme does not guarantee its suitability for use in an enzyme cocktail or as a designer cellulosome component. The true merit of improvement resides in its ultimate contribution to synergistic action, which can only be determined experimentally. The relevance of the mutated thermostable enzymes employed in this study as components in multienzyme systems has thus been confirmed using designer cellulosome technology. Enzyme integration via a thermostable scaffoldin is critical to the ultimate stability of the complex at higher temperatures. Engineering of thermostable cellulases and additional lignocellulosic enzymes may prove a determinant parameter for development of state-of-the-art designer cellulosomes for their employment in the conversion of cellulosic biomass to soluble sugars.Graphical abstractConversion of conventional designer cellulosomes into thermophilic designer cellulosomes.
authors: Department of Biomolecular Sciences, The Weizmann Institute of Science, 76100 Rehovot, Israel.
authors: Sarah
authors: S
authors: Moraïs
authors: Department of Biomolecular Sciences, The Weizmann Institute of Science, 76100 Rehovot, Israel.
authors: Johanna
authors: J
authors: Stern
authors: Department of Biomolecular Sciences, The Weizmann Institute of Science, 76100 Rehovot, Israel.
authors: Amaranta
authors: A
authors: Kahn
authors: Microbiology Group, Faculty of Biology, National and Kapodistrian University of Athens, Zografou Campus, 15784 Athens, Greece.
authors: Anastasia P
authors: AP
authors: Galanopoulou
authors: Department of Biomolecular Sciences, The Weizmann Institute of Science, 76100 Rehovot, Israel ; Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, P.O. Box 12, 76100 Rehovot, Israel.
authors: Shahar
authors: S
authors: Yoav
authors: Department of Biomolecular Sciences, The Weizmann Institute of Science, 76100 Rehovot, Israel.
authors: Melina
authors: M
authors: Shamshoum
authors: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125 USA.
authors: Matthew A
authors: MA
authors: Smith
authors: Microbiology Group, Faculty of Biology, National and Kapodistrian University of Athens, Zografou Campus, 15784 Athens, Greece.
authors: Dimitris G
authors: DG
authors: Hatzinikolaou
authors: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125 USA.
authors: Frances H
authors: FH
authors: Arnold
authors: Department of Biomolecular Sciences, The Weizmann Institute of Science, 76100 Rehovot, Israel ; Department of Biological Chemistry, The Weizmann Institute of Science, 76100 Rehovot, Israel.
authors: Edward A
authors: EA
authors: Bayer
keywords: Clostridium thermocellum; Designer cellulosomes; Multi-enzyme complex; Thermostable cellulases