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Optimal catalytic activity of endoglucanase Cel5D from the thermophilic anaerobic bacterium Caldicellulosiruptor bescii requires the presence of a carbohydrate-binding module of family 28, CbCBM28. The binding properties of CbСВМ28 with cello-, laminari-, xylo- and chito-oligosaccharides were studied by isothermal titration calorimetry. CbСВМ28 bound only cello-oligosaccharides comprising at least four glucose residues with binding constants of 2.5·10 4 and 2.2·10 6 M -1 for cellotetraose and cellohexaose, respectively. The interaction between CbСВМ28 and amorphous cellulose is best described by a two-binding-site model with the binding constants of 1.5·10 5 and 1.9·10 5 M -1 . In a competitive binding assay in the presence of a 10-fold excess of cellohexaose the binding constant of CbСВМ28 to amorphous cellulose was 1.9·10 5 M -1 . A two-binding-site model also better approximates the binding to Avicel with the binding constants of 8.3·10 5 and 3.2·10 4 M -1 ; while in the presence of cellohexaose, the binding is described by a single-binding-site model with the binding constant of 2.3·10 4 M -1 . With CbСВМ28 binding to bacterial crystalline cellulose with a constant of 7.4·10 4 M -1 , this is the first report of such a strong binding to crystalline cellulose for a module of family 28., (Copyright © 2017 Elsevier B.V. All rights reserved.)

A novel bacterial species was isolated from an industrial-scale biogas plant. The isolate Herbinix hemicellulosilytica T3/55(T) is able to degrade crystalline cellulose. Comparative 16S rRNA gene sequencing demonstrated that the isolate is closely related to environmental samples forming a hitherto unknown sub-cluster within the family Lachnospiraceae. The draft genome sequence of strain T3/55(T) was established and now provides the genetic basis for application of this microorganism in thermophilic degradation of lignocellulosic biomass., (Copyright © 2015. Published by Elsevier B.V.)

The bacterium Ruminiclostridium cellulosi DG5, a thermophilic, anaerobic member of the family Ruminococcaceae, was isolated from an industrial-scale biogas plant in Germany. It is able to grow efficiently on cellulose and cellodextrins. The whole genome sequence of R. cellulosi DG5 was established and now provides the genetic basis for biotechnological exploitation of genome features involved in thermophilic degradation of lignocellulosic biomass., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The heterogeneous plant biomass is efficiently decomposed by the interplay of a great number of different enzymes. The enzyme systems in cellulolytic bacteria have been investigated by sequencing and bioinformatic analysis of genomes from plant biomass degrading microorganisms with valuable insights into the variety of the involved enzymes. This broadened our understanding of the biochemical mechanisms of plant polymer degradation and made the enzymes applicable for modern biotechnology. A list of the truly cellulolytic bacteria described and the available genomic information was examined for proteins with cellulolytic and hemicellulolytic capability. The importance of the isolation, characterization and genomic sequencing of cellulolytic microorganisms and their usage for sustainable energy production from biomass and other residues, is emphasized., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Clostridium thermocellum is among the most prevalent of known anaerobic cellulolytic bacteria. In this study, genetic and phenotypic variations among C. thermocellum strains isolated from different biogas plants were determined and different genotyping methods were evaluated on these isolates. At least two C. thermocellum strains were isolated independently from each of nine different biogas plants via enrichment on cellulose. Various DNA-based genotyping methods such as ribotyping, RAPD (Random Amplified Polymorphic DNA) and VNTR (Variable Number of Tandem Repeats) were applied to these isolates. One novel approach - the amplification of unknown target sequences between copies of a previously discovered Random Inserted Mobile Element (RIME) - was also tested. The genotyping method with the highest discriminatory power was found to be the amplification of the sequences between the insertion elements, where isolates from each biogas plant yielded a different band pattern. Cellulolytic potentials, optimal growth conditions and substrate spectra of all isolates were characterized to help identify phenotypic variations. Irrespective of the genotyping method used, the isolates from each individual biogas plant always exhibited identical patterns. This is suggestive of a single C. thermocellum strain exhibiting dominance in each biogas plant. The genotypic groups reflect the results of the physiological characterization of the isolates like substrate diversity and cellulase activity. Conversely, strains isolated across a range of biogas plants differed in their genotyping results and physiological properties. Both strains isolated from one biogas plant had the best specific cellulose-degrading properties and might therefore achieve superior substrate utilization yields in biogas fermenters., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

The bacterium Clostridium thermocellum BC1, a thermophilic, anaerobic bacterium of the family Clostridiaceae, was isolated from a compost treatment site in Germany. It is able to grow efficiently on cellulose and cellodextrins. The draft genome sequence of C. thermocellum BC1 has been established and provides the genetic basis for application of this microorganism in thermophilic degradation of cellulosic biomass., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Artificial cellulase complexes active on crystalline cellulose were reconstituted in vitro from 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 a C. thermocellum cipA mutant. 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. The in 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 for in vitro complex reconstitution, which is an important step toward the creation of highly efficient engineered cellulases.

Mutants of Clostridium thermocellum that had lost the ability to adhere to microcrystalline cellulose were isolated. Six of them that showed diminished ability to depolymerize crystalline cellulose were selected. Size exclusion chromatography of the proteins from the culture supernatant revealed the loss of the supramolecular enzyme complex, the cellulosome. However, denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis resulted in extracellular protein patterns comparable to those of isolated cellulosomes, except for a missing CipA band. Sequencing of the six mutant cipA genes revealed a new insertion (IS) element, IS1447, belonging to the IS3 family. It was inserted into the cipA reading frame in four different locations: cohesin module 1, two different positions in the carbohydrate binding module, and cohesin module 3. The IS sequences were identical and consisted of a transposase gene and the inverted repeats IRR and IRS. The insertion resulted in an obviously nonspecific duplication of 3 base pairs within the target sequence. This lack of specificity allows transposition without the need of a defined target DNA sequence. Eighteen copies of IS1447 were identified in the genomic sequence of C. thermocellum ATCC 27405. At least one of them can be activated for transposition. Compared to the wild type, the mutant culture supernatant, with a completely defective CipA protein, showed equal specific hydrolytic activity against soluble beta-glucan but a 15-fold reduction in specific activity with crystalline cellulose. These results identify a genetic basis for the synergistic effect of complex formation on crystalline-cellulose degradation.

Cel9R, a major component in the cellulosome of Clostridium thermocellum, is one of the most prevalent beta-glucanases in the complex after Cel48S and Cel8A. The recombinant product of gene celR is optimally active at 78.5 degrees C on amorphous cellulose, carboxymethyl-cellulose, and barley beta-1,3-1,4-glucan. From amorphous cellulose it produces initially cellotetraose which is slowly degraded to glucose, cellobiose and cellotriose. This product pattern indicates a processive endoglucanase-mode which was corroborated by the initial and simultaneous production of new reducing ends in the soluble as well as in the insoluble fraction of amorphous cellulose. pNP-Cellopentaoside is degraded to cellotetraose and pNP-glucoside, suggesting cellotetraose release from the non-reducing end. The newly discovered Cel9R thus is a novel type of cellulase in the cellulosome of C. thermocellum: a processive endo-beta-1,4-glucanase producing cellotetraose as the primary hydrolysis product. The presence in the cellulosome and the hydrolytic mode of this cellotetraohydrolase has implications for our understanding of the in vivo conversion of cellulose by bacteria.

Clostridium thermocellum produces a great number of extracellular cellulases which are free or cellulosome-bound. The nucleotide sequence of a gene cluster containing the genes celI, celN and cseP was determined from C. thermocellum strain F7. Gene products Cel9I and Cel9N are structurally related enzymes having a glycosyl hydrolase family 9 and a carbohydrate-binding module (CBM3c), but show characteristic differences: Cel9I is a non-cellulosomal protein with an additional CBM (CBM3b), whereas Cel9N contains a cellulosomal dockerin module and no additional CBM. Although Cel9I is a processive endoglucanase, Cel9N is non-processive. Both enzymes hydrolyse phosphoric acid swollen cellulose, but the products of hydrolysis are different. The CseP protein encoded in the gene cluster is the first component attached to the cellulosomal scaffoldin for which no catalytic activity could be detected. It was shown to be present in the cellulosome. Its sequence is homologous to the spore-coat assembly protein CotH of Bacillus subtilis, suggesting a structural role of CseP in the cellulosome.

The sequence of the celO gene from Clostridium thermocellum F7 was determined. The gene product, cellulase CelO (Ct-Cel5F), had a modular structure consisting of a carbohydrate-binding module of the CBM3 family and a catalytic domain of the glycosyl hydrolase family 5. The presence of the dockerin module indicated that the enzyme was a component of the cellulosome complex. The thermostable recombinant gene product was active on cellodextrins, barley beta-glucan, carboxymethylcellulose and insoluble cellulose. Cellobiose was the only product released from amorphic and crystalline cellulose, cellotetraose and higher cello-oligosaccharides, identifying CelO as a cellobiohydrolase. The cleavage pattern of p-nitrophenyl beta-D-cellotetraoside, blockage of the hydrolysis of NaBH(4)-reduced cellopentaose and the reduction in substrate viscosity suggested activity from the reducing end in a processive mode after making random cuts. Binding to insoluble, i.e. amorphous, and crystalline cellulose was mediated by the carbohydrate-binding module CBM3b, with a preference for the crystalline substrate.

Cellulose degradation is a rare trait in bacteria. However, the truly cellulolytic bacteria are extremely efficient hydrolyzers of plant cell wall polysaccharides, especially those in thermophilic anaerobic ecosystems. Clostridium stercorarium, a thermophilic ubiquitous soil dweller, has a simple cellulose hydrolyzing enzyme system of only two cellulases. However, it seems to be better suited for the hydrolysis of a wide range of hemicelluloses. Clostridium thermocellum, an ubiquitous thermophilic gram-type positive bacterium, is one of the most successful cellulose degraders known. Its extracellular enzyme complex, the cellulosome, was prepared from C. thermocellum cultures grown on cellulose, cellobiose, barley β-1,3-1,4-glucan, or a mixture of xylan and cellulose. The single proteins were identified by peptide chromatography and MALDI-TOF-TOF. Eight cellulosomal proteins could be found in all eight preparations, 32 proteins occur in at least one preparation. A number of enzymatic components had not been identified previously. The proportion of components changes if C. thermocellum is grown on different substrates. Mutants of C. thermocellum, devoid of scaffoldin CipA, that now allow new types of experiments with in vitro cellulosome reassembly and a role in cellulose hydrolysis are described. The characteristics of these mutants provide strong evidence of the positive effect of complex (cellulosome) formation on hydrolysis of crystalline cellulose. [ABSTRACT FROM AUTHOR]

The genome of Clostridium thermocellum contains a number of genes for polysaccharide degradation-associated proteins that are not cellulosome bound. The list includes β-glucanases, glycosidases, chitinases, amylases and a xylanase. One of these ‘soluble’-enzyme genes codes for a second glycosyl hydrolase (GH)48 cellulase, Cel48Y, which was expressed in Escherichia coli and biochemically characterized. It is a cellobiohydrolyse with activity on native cellulose such as microcrystalline and bacterial cellulose, and low activity on carboxymethylcellulose. It is about 100 times as active on amorphic cellulose and mixed-linkage barley β-glucan compared with cellulase Cel9I. The enzyme Cel48Y shows a distinct synergism of 2.1 times with the noncellulosomal processive endoglucanase Cel9I on highly crystalline bacterial cellulose at a 17-fold excess of Cel48Y over Cel9I. These data show that C. thermocellum has, besides the cellulosome, the genes for a second cellulase system for the hydrolysis of crystalline cellulose that is not particle bound. [ABSTRACT FROM AUTHOR]

Abstract: Cel9R, a major component in the cellulosome of Clostridium thermocellum, is one of the most prevalent β-glucanases in the complex after Cel48S and Cel8A. The recombinant product of gene celR is optimally active at 78.5°C on amorphous cellulose, carboxymethyl-cellulose, and barley β-1,3-1,4-glucan. From amorphous cellulose it produces initially cellotetraose which is slowly degraded to glucose, cellobiose and cellotriose. This product pattern indicates a processive endoglucanase-mode which was corroborated by the initial and simultaneous production of new reducing ends in the soluble as well as in the insoluble fraction of amorphous cellulose. pNP-Cellopentaoside is degraded to cellotetraose and pNP-glucoside, suggesting cellotetraose release from the non-reducing end. The newly discovered Cel9R thus is a novel type of cellulase in the cellulosome of C. thermocellum: a processive endo-β-1,4-glucanase producing cellotetraose as the primary hydrolysis product. The presence in the cellulosome and the hydrolytic mode of this cellotetraohydrolase has implications for our understanding of the in vivo conversion of cellulose by bacteria. [Copyright &y& Elsevier]

Determines the structure and enzymatic activity of xyloglucanase Xgh74A and endoxylanase Xyn10D, which are components in the cellulosomes of cellulose-grown Clostridium thermocellum. Characteristics and functions of the enzymes; Indication that both contribute to the in vivo degradation of the hemicelluloses xyloglucan and xylan by the cellulosome.