Interplay between Clostridium thermocellum family 48 and family 9 cellulases in cellulosomal versus noncellulosomal states

Yael Vazana, Sarah Moraïs, Yoav Barak, Raphael Lamed, Edward A. Bayer

Research output: Contribution to journalArticlepeer-review

61 Scopus citations

Abstract

The anaerobic, thermophilic cellulolytic bacterium Clostridium thermocellum is known for its elaborate cellulosome complex, but it also produces a separate free cellulase system. Among the free enzymes, the noncellulosomal enzyme Cel9I is a processive endoglucanase whose sequence and architecture are very similar to those of the cellulosomal enzyme Cel9R; likewise, the noncellulosomal exoglucanase Cel48Y is analogous to the principal cellulosomal enzyme Cel48S. In this study we used the designer cellulosome approach to examine the interplay of prominent cellulosomal and noncellulosomal cellulases from C. thermocellum. Toward this end, we converted the cellulosomal enzymes to noncellulosomal chimeras by swapping the dockerin module of the cellulosomal enzymes with a carbohydrate-binding module from the free enzyme analogues and vice versa. This enabled us to study the importance of the targeting effect of the free enzymes due to their carbohydrate-binding module and the proximity effect for cellulases on the designer cellulosome. C. thermocellum is the only cellulosome-producing bacterium known to express two different glycoside hydrolase family 48 enzymes and thus the only bacterial system that can currently be used for such studies. The different activities with crystalline cellulose were examined, and the results demonstrated that the individual chimeric cellulases were essentially equivalent to the corresponding wild-type analogues. The wild-type cellulases displayed a synergism of about 1.5-fold; the cellulosomal pair acted synergistically when they were converted into free enzymes, whereas the free enzymes acted synergistically mainly in the wild-type state. The targeting effect was found to be the major factor responsible for the elevated activity observed for these specific enzyme combinations, whereas the proximity effect appeared to play a negligible role.

Original languageEnglish
Pages (from-to)3236-3243
Number of pages8
JournalApplied and Environmental Microbiology
Volume76
Issue number10
DOIs
StatePublished - 1 May 2010
Externally publishedYes

ASJC Scopus subject areas

  • Biotechnology
  • Food Science
  • Ecology
  • Applied Microbiology and Biotechnology

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