scholarly journals In VitroReconstitution of the Complete Clostridium thermocellum Cellulosome and Synergistic Activity on Crystalline Cellulose

2012 ◽  
Vol 78 (12) ◽  
pp. 4301-4307 ◽  
Author(s):  
Jan Krauss ◽  
Vladimir V. Zverlov ◽  
Wolfgang H. Schwarz
Keyword(s):  
Clostridium Thermocellum ◽  
Protein Complexes ◽  
Hydrolytic Activity ◽  
Crystalline Cellulose ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Synergistic Activity ◽  
Crystalline Substrate ◽  
Culture Supernatants

ABSTRACTArtificial cellulase complexes active on crystalline cellulose were reconstitutedin vitrofrom a native mix of cellulosomal enzymes and CipA scaffoldin. Enzymes containing dockerin modules for binding to the corresponding cohesin modules were prepared from culture supernatants of aC. thermocellum cipAmutant. They were reassociated to cellulosomes via dockerin-cohesin interaction. Recombinantly produced mini-CipA proteins with one to three cohesins either with or without the carbohydrate-binding module (CBM) and the complete CipA protein were used as the cellulosomal backbone. The binding between cohesins and dockerins occurred spontaneously. The hydrolytic activity against soluble and crystalline cellulosic compounds showed that the composition of the complex does not seem to be dependent on which CipA-derived cohesin was used for reconstitution. Binding did not seem to have an obvious local preference (equal binding to Coh1 and Coh6). The synergism on crystalline cellulose increased with an increasing number of cohesins in the scaffoldin. Thein vitro-formed complex showed a 12-fold synergism on the crystalline substrate (compared to the uncomplexed components). The activity of reconstituted cellulosomes with full-size CipA reached 80% of that of native cellulosomes. Complexation on the surface of nanoparticles retained the activity of protein complexes and enhanced their stability. Partial supplementation of the native cellulosome components with three selected recombinant cellulases enhanced the activity on crystalline cellulose and reached that of the native cellulosome. This opens possibilities forin vitrocomplex reconstitution, which is an important step toward the creation of highly efficient engineered cellulases.

scholarly journals Interplay between Clostridium thermocellum Family 48 and Family 9 Cellulases in Cellulosomal versus Noncellulosomal States

2010 ◽  
Vol 76 (10) ◽  
pp. 3236-3243 ◽  
Author(s):  
Yael Vazana ◽  
Sarah Moraïs ◽  
Yoav Barak ◽  
Raphael Lamed ◽  
Edward A. Bayer
Keyword(s):  
Proximity Effect ◽  
Clostridium Thermocellum ◽  
Crystalline Cellulose ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Cellulolytic Bacterium ◽  
Carbohydrate Binding Module ◽  
Wild Type ◽  
Designer Cellulosome ◽  
Targeting Effect

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.

scholarly journals Regulation of Adsorption Behavior of Carbohydrate-Binding Module Family 1 and Endo-β-1,4-Glucanase onto Crystalline Cellulose

2011 ◽  
Vol 58 (4) ◽  
pp. 133-138 ◽  
Author(s):  
Kouichi Nozaki ◽  
Hiroto Nishijima ◽  
Tsutomu Arai ◽  
Masahiro Mizuno ◽  
Nobuaki Sato ◽  
...  
Keyword(s):  
Adsorption Behavior ◽  
Crystalline Cellulose ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module

Influence of positioning of carbohydrate binding module on the activity of endoglucanase CelA of Clostridium thermocellum

2012 ◽  
Vol 161 (3) ◽  
pp. 206-212 ◽  
Author(s):  
Muhammad Sajjad ◽  
M. Imran Mahmood Khan ◽  
Rehan Zafar ◽  
Sajjad Ahmad ◽  
Umar H.K. Niazi ◽  
...  
Keyword(s):  
Clostridium Thermocellum ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module

The nature of the carbohydrate binding module determines the catalytic efficiency of xylanase Z of Clostridium thermocellum

2013 ◽  
Vol 168 (4) ◽  
pp. 403-408 ◽  
Author(s):  
Muhammad Imran M. Khan ◽  
Muhammad Sajjad ◽  
Saima Sadaf ◽  
Rehan Zafar ◽  
Umer H.K. Niazi ◽  
...  
Keyword(s):  
Clostridium Thermocellum ◽  
Catalytic Efficiency ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module

scholarly journals Heterologous Production, Assembly, and Secretion of a Minicellulosome by Clostridium acetobutylicum ATCC 824

2005 ◽  
Vol 71 (3) ◽  
pp. 1215-1222 ◽  
Author(s):  
Florence Mingardon ◽  
Stéphanie Perret ◽  
Anne Bélaïch ◽  
Chantal Tardif ◽  
Jean-Pierre Bélaïch ◽  
...  
Keyword(s):  
Clostridium Acetobutylicum ◽  
Recombinant Strain ◽  
Large Fraction ◽  
Heterologous Gene ◽  
Crystalline Cellulose ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Heterologous Production ◽  
Suitable Host ◽  
Clostridium Acetobutylicum Atcc 824

ABSTRACT The gene man5K encoding the mannanase Man5K from Clostridium cellulolyticum was cloned alone or as an operon with the gene cipC1 encoding a truncated scaffoldin (miniCipC1) of the same origin in the solventogenic Clostridium acetobutylicum. The expression of the heterologous gene(s) was under the control of a weakened thiolase promoter P thl . The recombinant strains of the solventogenic bacterium were both found to secrete active Man5K in the range of milligrams per liter. In the case of the strain expressing only man5K, a large fraction of the recombinant enzyme was truncated and lost the N-terminal dockerin domain, but it remained active towards galactomannan. When man5K was coexpressed with cipC1 in C. acetobutylicum, the recombinant strain secreted almost exclusively full-length mannanase, which bound to the scaffoldin miniCipC1, thus showing that complexation to the scaffoldin stabilized the enzyme. The secreted heterologous complex was found to be functional: it binds to crystalline cellulose via the carbohydrate binding module of the miniscaffoldin, and the complexed mannanase is active towards galactomannan. Taken together, these data show that C. acetobutylicum is a suitable host for the production, assembly, and secretion of heterologous minicellulosomes.

scholarly journals Molecular basis for the preferential recognition of β1,3‐1,4‐glucans by the family 11 carbohydrate‐binding module from Clostridium thermocellum

FEBS Journal ◽  
2019 ◽  
Vol 287 (13) ◽  
pp. 2723-2743 ◽  
Author(s):  
Diana O. Ribeiro ◽  
Aldino Viegas ◽  
Virgínia M. R. Pires ◽  
João Medeiros‐Silva ◽  
Pedro Bule ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Clostridium Thermocellum ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
The Family ◽  
Preferential Recognition
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