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At least 19 recordsLinked to original sources

PubMed2006

Degradation of cellulosome-produced cello-oligosaccharides by an extracellular non-cellulosomal beta-glucan glucohydrolase, BglA, from Clostridium cellulovorans.

Clostridium cellulovorans degrades cellulose efficiently to small oligosaccharides, which are used as an energy source. To characterize enzymes related to degrading small oligosaccharides, a gene was cloned for an extracellular non-cellulosomal beta-glucan glucohydrolase (BglA) classified as a family-1 glycosyl hydrolase in C. cellulovorans. Recombinant BglA (rBglA) had higher activity on long glucooligomers than on cellobiose. When cellulosomes and rBglA were incubated with cellulose, the oligosaccharides produced were degraded more effectively to cellobiose and glucose, than with cellulosomes alone, indicating that BglA facilitated the degradation of accessible cello-oligosaccharides produced from cellulose by C. cellulovorans cellulosomes. Thus, this is an example of an extracellular non-cellulosomal enzyme working in a cooperative manner with cellulosomes to degrade cellulose to sugars.

Carbohydrates↗

PubMed2005

Regulation of major cellulosomal endoglucanases of Clostridium thermocellum differs from that of a prominent cellulosomal xylanase.

The expression of scaffoldin-anchoring genes and one of the major processive endoglucanases (CelS) from the cellulosome of Clostridium thermocellum has been shown to be dependent on the growth rate. For the present work, we studied the gene regulation of selected cellulosomal endoglucanases and a major xylanase in order to examine the previously observed substrate-linked alterations in cellulosome composition. For this purpose, the transcript levels of genes encoding endoglucanases CelB, CelG, and CelD and the family 10 xylanase XynC were determined in batch cultures, grown on either cellobiose or cellulose, and in carbon-limited continuous cultures at different dilution rates. Under all conditions tested, the transcript levels of celB and celG were at least 10-fold higher than that of celD. Like the major processive endoglucanase CelS, the transcript levels of these endoglucanase genes were also dependent on the growth rate. Thus, at a rate of 0.04 h(-1), the levels of celB, celG, and celD were threefold higher than those obtained in cultures grown at maximal rates (0.35 h(-1)) on cellobiose. In contrast, no clear correlation was observed between the transcript level of xynC and the growth rate-the levels remained relatively high, fluctuating between 30 and 50 transcripts per cell. The results suggest that the regulation of C. thermocellum endoglucanases is similar to that of the processive endoglucanase celS but differs from that of a major cellulosomal xylanase in that expression of the latter enzyme is independent of the growth rate.

Cellobiose↗

PubMed2004

Towards designer cellulosomes in Clostridia: mannanase enrichment of the cellulosomes produced by Clostridium cellulolyticum.

The man5K gene of Clostridium cellulolyticum was cloned and overexpressed in Escherichia coli. This gene encodes a 424-amino-acid preprotein composed of an N-terminal leader peptide, followed by a dockerin module and a C-terminal catalytic module belonging to family 5 of the glycosyl hydrolases. Mature Man5K displays 62% identity with ManA from Clostridium cellulovorans. Two forms of the protein were purified from E. coli; one form corresponds to the full-length enzyme (45 kDa), and a truncated form (39 kDa) lacks the N-terminal dockerin module. Both forms exhibit the same typical family 5 mannanase substrate preference; they are very active with the galactomannan locust bean gum, and the more galacto-substituted guar gum molecules are degraded less. The truncated form, however, displays fourfold-higher activity with galactomannans than the full-length enzyme. Man5K was successfully overproduced in C. cellulolyticum by using expression vectors. The trans-produced protein was found to be incorporated into the cellulosomes and became one of the major enzymatic components. Modified cellulosomes displayed 20-fold-higher specific activities than control fractions on galactomannan substrates, whereas the specific activity on crystalline cellulose was reduced by 20%. This work clearly showed that the composition of the cellulosomes is obviously regulated by the relative amounts of the enzymes produced and that this composition can be engineered in clostridia by structural gene cloning.

Amino Acid Sequence↗

PubMed1992

Involvement of separate domains of the cellulosomal protein S1 of Clostridium thermocellum in binding to cellulose and in anchoring of catalytic subunits to the cellulosome.

Fragments of the 250 kDa S1 subunit of the Clostridium thermocellum cellulosome were obtained by protease-induced or spontaneous degradation. All detectable fragments, down to a mass of about 30 kDa, retained the ability to bind to 125I-labelled endoglucanase CelD, one of the catalytic subunits of the cellulosome. Several fragments were able to bind both to cellulose and to CelD. However, some fragments that could still bind to CelD did not have the ability to bind to cellulose. Therefore, S1, a putative scaffolding protein of the cellulosome, is likely to carry two separate types of domains, one of which binds to cellulose, while the other type binds to the various catalytic subunits of the complex.

Bacterial Proteins↗

PubMed1998

Synergistic interaction of the cellulosome integrating protein (CipA) from Clostridium thermocellum with a cellulosomal endoglucanase.

Activity of a cellulosomal endoglucanase (endoglucanase E; EGE) from Clostridium thermocellum against two crystalline forms of cellulose was enhanced by combination with the cellulosome integrating protein (CipA), but CipA did not enhance EGE activity against amorphous cellulose, even though it was able to bind to it. Similarly, CipA added in trans to genetically truncated EGE that was unable to combine with it nevertheless enhanced EGE activity against crystalline cellulose. These results indicate that the CipA cellulose binding domain does not mediate an increase in activity solely by bringing the catalytic subunits of the cellulosome complex into intimate contact with the substrate.

Bacterial Proteins↗

PubMed1993

Organization of a Clostridium thermocellum gene cluster encoding the cellulosomal scaffolding protein CipA and a protein possibly involved in attachment of the cellulosome to the cell surface.

The nucleotide sequence was determined for a 9.4-kb region of Clostridium thermocellum DNA extending from the 3' end of the gene (now termed cipA), encoding the S1/SL component of the cellulosome. Three open reading frames (ORFs) belonging to two operons were detected. They encoded polypeptides of 1,664, 688, and 447 residues, termed ORF1p, ORF2p, and ORF3p, respectively. The COOH-terminal regions of the three polypeptides were highly similar and contained three reiterated segments of 60 to 70 residues each. Similar segments have been found at the NH2 terminus of the S-layer proteins of Bacillus brevis and Acetogenium kivui, suggesting that ORF1p, ORF2p, and ORF3p might also be located on the cell surface. Otherwise, the sequence of ORF1p and ORF2p gave little clue concerning their potential function. However, the NH2-terminal region of ORF3p was similar to the reiterated domains previously identified in CipA as receptors involved in binding the duplicated segment of 22 amino acids present in catalytic subunits of the cellulosome. Indeed, it was found previously that ORF3p binds 125I-labeled endoglucanase CelD containing the duplicated segment (T. Fujino, P. Béguin, and J.-P. Aubert, FEMS Microbiol. Lett. 94:165-170, 1992). These findings suggest that ORF3p might serve as an anchoring factor for the cellulosome on the cell surface by binding the duplicated segment that is present at the COOH end of CipA.

Amino Acid Sequence↗

PubMed2002

Thermostabilization of cellulosomal endoglucanase EngB from Clostridium cellulovorans by in vitro DNA recombination with non-cellulosomal endoglucanase EngD.

Enhancement of enzyme thermostability by protein engineering gives us information about the thermostabilization mechanism as well as advantages for industrial use of enzymes. In this study, we enhanced the thermostability of endoglucanase EngB, one component of the cellulase complex (cellulosome) from Clostridium cellulovorans, by the directed evolution technique. The library was constructed by in vitro recombination of the genes for EngB and non-cellulosomal cellulase EngD, based on the fact that the catalytic domains of both cellulases were highly homologous. To obtain thermostable clones without loss of activity, the library was screened by a combination of activity and thermostability screening. We obtained three mutants out of 8000 selected clones that showed significantly higher thermostability than those of EngB and EngD without compromising their endoglucanase activities. One of the mutants possessed a sevenfold higher thermostability than EngB. The possible mechanisms of thermostabilization are discussed.

Amino Acid Sequence↗

PubMed2001

The Clostridium cellulovorans cellulosome: an enzyme complex with plant cell wall degrading activity.

Cellulose comprises a major portion of biomass on the earth, and the turnover of this material contributes to the CO2 cycle. Cellulases, which play a major role in the turnover of cellulosic materials, have been found either as free enzymes that work synergistically, or as an enzyme complex called the cellulosome. This review summarizes some of the general properties of cellulosomes, and more specifically, the properties of the Clostridium cellulovorans cellulosome. The C cellulovorans cellulosome is an extracellular enzyme complex with a molecular weight of about 1 x 10(6), and is comprised of at least ten subunits. The major subunit is the scaffolding protein CbpA, with a molecular weight of 189,000. This nonenzymatic subunit contains a cellulose binding domain (CBD) that binds the cellulosome to the substrate, nine conserved cohesins or enzyme binding domains, and four conserved surface layer homologous (SLH) domains. It is postulated that the SLH domains help to bind the cellulosome to the cell surface. The cellulosomal enzymes include cellulases (family 5 and 9 endoglucanases and a family 48 exoglucanase), a mannanase, a xylanase, and a pectate lyase. The cellulosome is capable of converting Arabidopsis and tobacco plant cells to protoplasts. One of the endoglucanases, EngE, contains three tandemly repeated SLHs at its N-terminus, and therefore appears capable of binding to the scaffolding protein CbpA as well as to the cell surface. Cellulosomes can attack crystalline cellulose, but the free cellulosomal enzymes can attack only soluble and amorphous celluloses. Nine genes for the cellulosome are found in a gene cluster cbpA-exgS-engH-engK-hbpA-engL-manA-engM-engN. Other cellulosomal genes such as engB, engE, and engY are not linked to the major gene cluster or to each other. By determining the structure and function of the cellulosome, we hope to increase the efficiency of the cellulosome by genetic engineering techniques.

Amino Acid Sequence↗

PubMed1999

Digestion of crystalline cellulose substrates by the clostridium thermocellum cellulosome: structural and morphological aspects.

The action of cellulosomes from Clostridium thermocellum on model cellulose microfibrils from Acetobacter xylinum and cellulose microcrystals from Valonia ventricosa was investigated. The biodegradation of these substrates was followed by transmission electron microscopy, Fourier-transform IR spectroscopy and X-ray diffraction analysis, as a function of the extent of degradation. The cellulosomes were very effective in catalysing the complete digestion of bacterial cellulose, but the total degradation of Valonia microcrystals was achieved more slowly. Ultrastructural observations during the digestion process suggested that the rapid degradation of bacterial cellulose was the result of a very efficient synergistic action of the various enzymic components that are attached to the scaffolding protein of the cellulosomes. The degraded Valonia sample assumed various shapes, ranging from thinned-down microcrystals to crystals where one end was pointed and the other intact. This complexity may be correlated with the multi-enzyme content of the cellulosomes and possibly to a diversity of the cellulosome composition within a given batch. Another aspect of the digestion of model celluloses by cellulosomes is the relative invariability of their crystallinity, together with their Ialpha/Ibeta composition throughout the degradation process. Comparison of the action of cellulosomes with that of fungal enzymes indicated that the degradation of cellulose crystals by cellulosomes occurred with only limited levels of processivity, in contrast with the observations reported for fungal enzymes. The findings were consistent with a mechanism whereby initial attack by a cellulosome of an individual cellulose crystal results in its 'commitment' towards complete degradation.

Acetobacter↗

PubMed2005

Effect of carbon source on the cellulosomal subpopulations of Clostridium cellulovorans.

Clostridium cellulovorans produces a cellulase enzyme complex called the cellulosome. When cells were grown on different carbon substrates such as Avicel, pectin, xylan, or a mixture of all three, the subunit composition of the cellulosomal subpopulations and their enzymic activities varied significantly. Fractionation of the cellulosomes (7-11 fractions) indicated that the cellulosome population was heterogeneous, although the composition of the scaffolding protein CbpA, endoglucanase EngE and cellobiohydrolase ExgS was relatively constant. One of the cellulosomal fractions with the greatest endoglucanase activity also showed the highest or second highest cellulase activity under all growth conditions tested. The cellulosomal fractions produced from cells grown on a mixture of carbon substrates showed the greatest cellulase activity and contained CbpA, EngE/EngK, ExgS/EngH and EngL. High xylanase activity in cellulose, pectin and mixed carbon-grown cells was detected with a specific cellulosomal fraction which had relatively larger amounts of XynB, XynA and unknown proteins (35-45 kDa). These results in toto indicate that the assembly of cellulosomes occurs in a non-random fashion.

Blotting, Western↗

PubMed2004

Hydrophilic domains of scaffolding protein CbpA promote glycosyl hydrolase activity and localization of cellulosomes to the cell surface of Clostridium cellulovorans.

CbpA, the scaffolding protein of Clostridium cellulovorans cellulosomes, possesses one family 3 cellulose binding domain, nine cohesin domains, and four hydrophilic domains (HLDs). Among the three types of domains, the function of the HLDs is still unknown. We proposed previously that the HLDs of CbpA play a role in attaching the cellulosome to the cell surface, since they showed some homology to the surface layer homology domains of EngE. Several recombinant proteins with HLDs (rHLDs) and recombinant EngE (rEngE) were examined to determine their binding to the C. cellulovorans cell wall fraction. Tandemly linked rHLDs showed higher affinity for the cell wall than individual rHLDs showed. EngE was shown to have a higher affinity for cell walls than rHLDs have. C. cellulovorans native cellulosomes were found to have higher affinity for cell walls than rHLDs have. When immunoblot analysis was carried out with the native cellulosome fraction bound to cell wall fragments, the presence of EngE was also confirmed, suggesting that the mechanism anchoring CbpA to the C. cellulovorans cell surface was mediated through EngE and that the HLDs play a secondary role in the attachment of the cellulosome to the cell surface. During a study of the role of HLDs on cellulose degradation, the mini-cellulosome complexes with HLDs degraded cellulose more efficiently than complexes without HLDs degraded cellulose. The rHLDs also showed binding affinity for crystalline cellulose and carboxymethyl cellulose. These results suggest that the CbpA HLDs play a major role and a minor role in C. cellulovorans cellulosomes. The primary role increases cellulose degradation activity by binding the cellulosome complex to the cellulose substrate; secondarily, HLDs aid the binding of the CbpA/cellulosome to the C. cellulovorans cell surface.

Amino Acid Sequence↗

PubMed2003

beta-Glucosidase in cellulosome of the anaerobic fungus Piromyces sp. strain E2 is a family 3 glycoside hydrolase.

The cellulosomes of anaerobic fungi convert crystalline cellulose solely into glucose, in contrast with bacterial cellulosomes which produce cellobiose. Previously, a beta-glucosidase was identified in the cellulosome of Piromyces sp. strain E2 by zymogram analysis, which represented approx. 25% of the extracellular beta-glucosidase activity. To identify the component in the fungal cellulosome responsible for the beta-glucosidase activity, immunoscreening with anti-cellulosome antibodies was used to isolate the corresponding gene. A 2737 bp immunoclone was isolated from a cDNA library. The clone encoded an extracellular protein containing a eukaryotic family 3 glycoside hydrolase domain homologue and was therefore named cel3A. The C-terminal end of the encoded Cel3A protein consisted of an auxiliary domain and three fungal dockerins, typical for cellulosome components. The Cel3A catalytic domain was expressed in Escherichia coli BL21 and purified. Biochemical analyses of the recombinant protein showed that the Cel3A catalytic domain was specific for beta-glucosidic bonds and functioned as an exoglucohydrolase on soluble substrates as well as cellulose. Comparison of the apparent K (m) and K (i) values of heterologous Cel3A and the fungal cellulosome for p -nitrophenyl-beta-D-glucopyranoside and D-glucono-1,5-delta-lactone respectively indicated that cel3A encodes the beta-glucosidase activity of the Piromyces sp. strain E2 cellulosome.

Amino Acid Sequence↗

PubMed2005

Action of designer cellulosomes on homogeneous versus complex substrates: controlled incorporation of three distinct enzymes into a defined trifunctional scaffoldin.

In recent work, we reported the self-assembly of a comprehensive set of defined "bifunctional" chimeric cellulosomes. Each complex contained the following: (i) a chimeric scaffoldin possessing a cellulose-binding module and two cohesins of divergent specificity and (ii) two cellulases, each bearing a dockerin complementary to one of the divergent cohesins. This approach allowed the controlled integration of desired enzymes into a multiprotein complex of predetermined stoichiometry and topology. The observed enhanced synergy on recalcitrant substrates by the bifunctional designer cellulosomes was ascribed to two major factors: substrate targeting and proximity of the two catalytic components. In the present work, the capacity of the previously described chimeric cellulosomes was amplified by developing a third divergent cohesin-dockerin device. The resultant trifunctional designer cellulosomes were assayed on homogeneous and complex substrates (microcrystalline cellulose and straw, respectively) and found to be considerably more active than the corresponding free enzyme or bifunctional systems. The results indicate that the synergy between two prominent cellulosomal enzymes (from the family-48 and -9 glycoside hydrolases) plays a crucial role during the degradation of cellulose by cellulosomes and that one dominant family-48 processive endoglucanase per complex is sufficient to achieve optimal levels of synergistic activity. Furthermore cooperation within a cellulosome chimera between cellulases and a hemicellulase from different microorganisms was achieved, leading to a trifunctional complex with enhanced activity on a complex substrate.

Bacterial Proteins↗

PubMed2006

Effect of sulfur-containing compounds on Bacillus cellulosome-associated 'CMCase' and 'Avicelase' activities.

The isolation of cellulosomes from clostridial sources has been extensively studied; however, the isolation of cellulosomes from facultative soil anaerobes of the family Bacillaceae is not as well characterized. The Bacillus cellulosome (celluloxylanosome) essentially consists of two complex components: C-I and C-II. This multi-component complex enables Bacillus to degrade a variety of carbonaceous compounds as it is composed of several enzymes, such as cellulases, xylanases and other degradative enzymes. The cellulosomal cellulases from Bacillus megaterium were purified using cellulose affinity chromatography, followed by Sepharose 4B gel filtration chromatography. The objective of this investigation was to establish the effect of sulfate and sulfide on cellulosomal 'cellulase' activity. An increase in sulfide concentration led to a general enhancement of cellulosomal-associated cellulolytic activity, whereas an increase in sulfate concentration resulted in an inhibition of the cellulosome-associated cellulolytic activity.

Bacillus↗

PubMed1996

Dissociation of the cellulosome of Clostridium thermocellum in the presence of ethylenediaminetetraacetic acid occurs with the formation of trucated polypeptides.

The cellulosome of Clostridium thermocellum JW20 consists of 14-26 different polypeptides as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The intact cellulosomes hydrolyzes crystalline cellulose in the presence of Ca2+ and thiols. This activity is inhibited by ethylenediaminetetraacetic acid (EDTA). Ca is incorporated into the cellulosome and tightly bound as demonstrated using 45Ca added to the growth medium. Upon incubation in 50 mM Tris (pH 7.5), 0.1 M NaCl, and 5 mM EDTA at 37 degrees C, Ca is released from the cellulosome, which disintegrates into polypeptides. The SDS-PAGE pattern of cellulosomal polypeptides is remarkably different after the EDTA treatment when compared to this pattern of untreated cellulosomes. Polypeptide bands corresponding to molecular masses of 160, 98, 76, 91, and 54 kDa disappear, and new bands of masses 150, 132, 91, 71, 57, and 46 kDa appear. N-terminal analyses of the 98, 76, 91, and 71 kDa polypeptides show that the 91 and 71 kDa polypeptides are truncated products of the 98 and 76 kDa polypeptides, respectively. The 76 and 71 kDa polypeptides correspond to CelS [Wang, W. K., Kruus K., & Wu, J. H. D. (1993) J. Bacteriol. 175, 1293-1302]. The 71 kDa polypeptide is formed from the 76 kDa polypeptide during the EDTA treatment, by a cleavage that occurs at asparagine residue 681. It involves the removal of 60 amino acid residues from the C-terminal end. All catalytic subunits so far characterized contain an asparagine residue corresponding to residue 681 of CelS. This residue is part of the conserved duplicated region found in catalytically active subunits, and it is postulated that several of these subunits also are truncated by the EDTA treatment. The polypeptides truncated by the EDTA treatment reduced Ca binding capacities compared to their native subunits, indicating a Ca-binding site within the conserved duplicated region. This region has been implicated in the binding of the catalytic peptides to the scaffolding polypeptide CipA, which is a structural protein of the cellulosome and has a strong affinity for calcium.

Amino Acid Sequence↗

PubMed2002

Characterization of the cellulolytic complex (cellulosome) of Clostridium acetobutylicum.

A large cellulosomal gene cluster was identified in the recently sequenced genome of Clostridium acetobutylicum ATCC 824. Sequence analysis revealed that this cluster contains the genes for the scaffolding protein CipA, the processive endocellulase Cel48A, several endoglucanases of families 5 and 9, the mannanase Man5G, and a hydrophobic protein, OrfXp. Surprisingly, genetic organization of this large cluster is very similar to that of Clostridium cellulolyticum, the model of mesophilic clostridial cellulosomes. As C. acetobutylicum is unable to grow on cellulosic substrates, the existence of a cellulosomal gene cluster in the genome raises questions about its expression, function and evolution. Biochemical evidence for the expression of a cellulosomal protein complex was investigated. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, N-terminal sequencing and Western blotting with antibodies against specific components of the C. cellulolyticum cellulosome suggest that at least four major cellulosomal proteins are present. In addition, despite the fact that no cellulolytic activities were detected, we report here the evidence for the production of a high molecular mass cellulosomal complex in C. acetobutylicum.

Amino Acid Sequence↗

PubMed1994

Cellulose promotes extracellular assembly of Clostridium cellulovorans cellulosomes.

Cellulosome synthesis by Clostridium cellulovorans was investigated by growing the cells in media containing different carbon sources. Supernatant from cells grown with cellobiose contained no cellulosomes and only the free forms of cellulosomal major subunits CbpA, P100, and P70 and the minor subunits with enzymatic activity. Supernatant from cells grown on pebble-milled cellulose and Avicel contained cellulosomes capable of degrading crystalline cellulose. Supernatants from cells grown with cellobiose, pebble-milled cellulose, and Avicel contained about the same amount of carboxymethyl cellulase activity. Although the supernatant from the medium containing cellobiose did not initially contain active cellulosomes, the addition of crystalline cellulose to the cell-free supernatant fraction converted the free major forms to cellulosomes with the ability to degrade crystalline cellulose. The binding of P100 and P70 to crystalline cellulose was dependent on their attachment to the endoglucanase-binding domains of CbpA. These data strongly indicate that crystalline cellulose promotes cellulosome assembly.

Bacterial Proteins↗

PubMed2004

Regulation of expression of cellulosomes and noncellulosomal (hemi)cellulolytic enzymes in Clostridium cellulovorans during growth on different carbon sources.

Cellulosomes and noncellulosomal (hemi)cellulolytic enzymes are produced by Clostridium cellulovorans to degrade plant cell walls. To understand their synergistic relationship, changes in mRNA and protein expression in cellulosomes and noncellulosomal (hemi)cellulolytic enzymes (hereafter called noncellulosomal enzymes) of cultures grown on cellobiose, cellulose, pectin, xylan, and corn fiber or mixtures thereof were examined. Cellulase expression, favored particularly by the presence of Avicel, was found with all substrates. Comparison of cellulosome and noncellulosomal enzymes showed that expression profiles were strongly affected by the carbon source. High xylanase or pectate lyase expression was observed when C. cellulovorans was grown on xylan or pectin, respectively. Mixed carbon substrates (cellulose-pectin-xylan mixture or corn fiber) induced a wider variety of enzymes than a single carbon source, such as cellobiose, pectin, or xylan. Cellulosomal proteome profiles were more affected by the carbon source than the noncellulosomal enzymes. Transcription and protein analyses revealed that cellulosomes and noncellulosomal enzymes were expressed simultaneously on mixed carbon sources, but their degree of inducibility varied when the substrate was either cellulose or cellobiose. Cellulosomes and noncellulosomal enzymes had synergistic activity on various carbon substrates. These results indicated that expression of plant cell wall-degrading enzymes is highly influenced by the available carbon source and that synergy between cellulosomes and noncellulosomal enzymes contribute to plant cell wall degradation.

Cellulase↗