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 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 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

scholarly journals Cloning and Characterization of Two Xyloglucanases from Paenibacillus sp. Strain KM21

2005 ◽  
Vol 71 (12) ◽  
pp. 7670-7678 ◽  
Author(s):  
Katsuro Yaoi ◽  
Tomonori Nakai ◽  
Yoshiro Kameda ◽  
Ayako Hiyoshi ◽  
Yasushi Mitsuishi
Keyword(s):  
Glycoside Hydrolase ◽  
Amino Acid Sequences ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
C Terminus ◽  
Paenibacillus Sp ◽  
Genes Encoding ◽  
Molecular Masses

ABSTRACT Two xyloglucan-specific endo-β-1,4-glucanases (xyloglucanases [XEGs]), XEG5 and XEG74, with molecular masses of 40 kDa and 105 kDa, respectively, were isolated from the gram-positive bacterium Paenibacillus sp. strain KM21, which degrades tamarind seed xyloglucan. The genes encoding these XEGs were cloned and sequenced. Based on their amino acid sequences, the catalytic domains of XEG5 and XEG74 were classified in the glycoside hydrolase families 5 and 74, respectively. XEG5 is the first xyloglucanase belonging to glycoside hydrolase family 5. XEG5 lacks a carbohydrate-binding module, while XEG74 has an X2 module and a family 3 type carbohydrate-binding module at its C terminus. The two XEGs were expressed in Escherichia coli, and recombinant forms of the enzymes were purified and characterized. Both XEGs had endoglucanase active only toward xyloglucan and not toward Avicel, carboxymethylcellulose, barley β-1,3/1,4-glucan, or xylan. XEG5 is a typical endo-type enzyme that randomly cleaves the xyloglucan main chain, while XEG74 has dual endo- and exo-mode activities or processive endo-mode activity. XEG5 digested the xyloglucan oligosaccharide XXXGXXXG to produce XXXG, whereas XEG74 digestion of XXXGXXXG resulted in XXX, XXXG, and GXXXG, suggesting that this enzyme cleaves the glycosidic bond of unbranched Glc residues. Analyses using various oligosaccharide structures revealed that unique structures of xyloglucan oligosaccharides can be prepared with XEG74.

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

Glycosylation by Pichia pastoris decreases the affinity of a family 2a carbohydrate-binding module from Cellulomonas fimi: a functional and mutational analysis

2001 ◽  
Vol 358 (2) ◽  
pp. 423-430 ◽  
Author(s):  
Alisdair B. BORASTON ◽  
R. Antony J. WARREN ◽  
Douglas G. KILBURN
Keyword(s):  
Pichia Pastoris ◽  
Association Constant ◽  
Mutational Analysis ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Wild Type ◽  
Cellulomonas Fimi ◽  
E Coli ◽  
Glycosylation Sites ◽  
High Mannose

When produced by Pichia pastoris, three of the five Asn-Xaa-Ser/Thr sequences (corresponding to Asn-24, Asn-73 and Asn-87) in the carbohydrate-binding module CBM2a of xylanase 10A from Cellulomonas fimi are glycosylated. The glycans are of the high-mannose type, ranging in size from GlcNAc2Man8 to GlcNAc2Man14. The N-linked glycans block the binding of CBM2a to cellulose. Analysis of mutants of CBM2a shows that glycans on Asn-24 decrease the association constant (Ka) for the binding of CBM2a to bacterial microcrystalline cellulose approx. 10-fold, whereas glycans on Asn-87 destroy binding. The Ka of a mutant of CBM2a lacking all three N-linked glycosylation sites is the same when the polypeptide is produced by either Escherichia coli or P. pastoris and is approx. half that of wild-type CBM2a produced by E. coli.

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 Global View of the Clostridium thermocellum Cellulosome Revealed by Quantitative Proteomic Analysis

2007 ◽  
Vol 189 (19) ◽  
pp. 6787-6795 ◽  
Author(s):  
Nicholas D. Gold ◽  
Vincent J. J. Martin
Keyword(s):  
Catabolite Repression ◽  
Clostridium Thermocellum ◽  
Transcriptional Control ◽  
Isotope Labeling ◽  
Expression Patterns ◽  
Peptide Sequencing ◽  
Glycoside Hydrolase Family ◽  
Cellulolytic Bacterium ◽  
Ionization Mass ◽  
Type I

ABSTRACT A metabolic isotope-labeling strategy was used in conjunction with nano-liquid chromatography-electrospray ionization mass spectrometry peptide sequencing to assess quantitative alterations in the expression patterns of subunits within cellulosomes of the cellulolytic bacterium Clostridium thermocellum, grown on either cellulose or cellobiose. In total, 41 cellulosomal proteins were detected, including 36 type I dockerin-containing proteins, which count among them all but three of the known docking components and 16 new subunits. All differential expression data were normalized to the scaffoldin CipA such that protein per cellulosome was compared for growth between the two substrates. Proteins that exhibited higher expression in cellulosomes from cellulose-grown cells than in cellobiose-grown cells were the cell surface anchor protein OlpB, exoglucanases CelS and CelK, and the glycoside hydrolase family 9 (GH9) endoglucanase CelJ. Conversely, lower expression in cellulosomes from cells grown on cellulose than on cellobiose was observed for the GH8 endoglucanase CelA; GH5 endoglucanases CelB, CelE, CelG; and hemicellulases XynA, XynC, XynZ, and XghA. GH9 cellulases were the most abundant group of enzymes per CipA when cells were grown on cellulose, while hemicellulases were the most abundant group on cellobiose. The results support the existing theory that expression of scaffoldin-related proteins is coordinately regulated by a catabolite repression type of mechanism, as well as the prior observation that xylanase expression is subject to a growth rate-independent type of regulation. However, concerning transcriptional control of cellulases, which had also been previously shown to be subject to catabolite repression, a novel distinction was observed with respect to endoglucanases.

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