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PubMed1998

Cloning, sequencing, and expression of an endoglucanase gene from the rumen anaerobic fungus Neocallimastix frontalis MCH3.

A cDNA clone encoding an endo-1,4-beta-glucanase from a rumen fungus, Neocallimastix frontalis MCH3, was isolated. The nucleotide sequence showed that the gene, celA, encoded a multidomain enzyme containing a family 5 catalytic domain and a reiterated sequence that is involved in the association of a multienzyme complex, the cellulosome. The enzyme expressed in Escherichia coli showed the highest activity against carboxymethylcellulose at 40 degrees C and pH 8.5.

Amino Acid Sequence↗

PubMed2003

A novel cellulolytic, anaerobic, and thermophilic bacterium, Moorella sp. strain F21.

A cellulolytic and thermophilic anaerobe was isolated from soil. This bacterium made a halo on a roll-tube culture containing Avicel. Analysis of the PCR-based 16S rRNA gene sequence showed that the bacterium was closely related to Moorella thermoacetica. Scanning electron microscopy showed the bacterium is a rod and has no protuberant structure on the surface of cells growing on cellulose, suggesting that this strain is a non-cellulosomal cellulolytic bacterium. Carboxymethyl cellulase and xylanase activities were detected in the culture broth. A major fermentation product from ball-milled cellulose was acetate. This strain has a potential to convert cellulosic biomass to acetate, directly.

Bacteria, Anaerobic↗

PubMed2005

Functions of family-22 carbohydrate-binding module in Clostridium thermocellum Xyn10C.

Clostridium thermocellum xylanase Xyn10C (formerly XynC) is a modular enzyme, comprising a family-22 carbohydrate-binding module (CBM), a family-10 catalytic module of the glycoside hydrolases, and a dockerin module responsible for cellulosome assembly consecutively from the N-terminus. To study the functions of the CBM, truncated derivatives of Xyn10C were constructed: a recombinant catalytic module polypeptide (rCM), a family-22 CBM polypeptide (rCBM), and a polypeptide composed of the family-22 CBM and CM (rCBM-CM). The recombinant proteins were characterized by enzyme and binding assays. Although the catalytic activity of rCBM-CM toward insoluble xylan was four times higher than that of rCM toward the same substrate, removal of the CBM did not severely affect catalytic activity toward soluble xylan or beta-1,3-1,4-glucan. rCBM showed an affinity for amorphous celluloses and insoluble and soluble xylan in qualitative binding assays. The optimum temperature of rCBM-CM was 80 degrees C and that of rCM was 60 degrees C. These results indicate that the family-22 CBM of C. thermocellum Xyn10C not only was responsible for the binding of the enzyme to the substrates, but also contributes to the stability of the CM in the presence of the substrate at high temperatures.

Base Sequence↗

PubMed2003

CelF of Orpinomyces PC-2 has an intron and encodes a cellulase (CelF) containing a carbohydrate-binding module.

A cDNA, designated celF, encoding a cellulase (CelF) was isolated from the anaerobic fungus Orpinomyces PC-2. The open reading frame contains regions coding for a signal peptide, a carbohydrate-binding module (CBM), a linker, and a catalytic domain. The catalytic domain was homologous to those of CelA and CelC of the same fungus and to that of the Neocallimastix patriciarum CELA, but CelF lacks a docking domain, characteristic for enzymes of cellulosomes. It was also homologous to the cellobiohydrolase IIs and endoglucanases of aerobic organisms. The gene has a 111-bp intron, located within the CBM-coding region. Some biochemical properties of the purified recombinant enzyme are described.

Base Sequence↗

PubMed2004

Cross bridging proteins in nature and their utilization in bio- and nanotechnology.

Cross-linking and cross-bridging are highly versatile methods of creating composite protein structures with desired mechanical properties such as deformation endurance, elasticity, extensibility, and stability under intensive and repetitive sheering forces. Cross-linking and cross-bridging are distinguished by the bonds that hold the structural components together. Cross-linking implies a covalent association, whereas cross-bridging depends on biological recognition, in which hydrogen bonding, ionic, and hydrophobic interactions predominate. Cross-bridged structures are found in all living systems. Cytoskeletal interaction, cell invasion by pathogens, fertilization, and cellulosomal degradation of cellulose are all examples of biological processes in which cross-bridging proteins play a key role. This article will review the different types of biological cross-bridging proteins that are known and discuss their emerging nano- and biotechnological applications.

Animals↗

PubMed1999

Homologous xylanases from Clostridium thermocellum: evidence for bi-functional activity, synergism between xylanase catalytic modules and the presence of xylan-binding domains in enzyme complexes.

Clostridium thermocellum produces a consortium of plant-cell-wall hydrolases that form a cell-bound multi-enzyme complex called the cellulosome. In the present study two similar xylanase genes, xynU and xynV, were cloned from C. thermocellum strain YS and sequenced. The deduced primary structures of both xylanases, xylanase U (XylU) and xylanase V (XylV), were homologous with the previously characterized xylanases from C. thermocellum strain F1. Truncated derivatives of XylV were produced and their biochemical properties were characterized. The xylanases were shown to be remarkably thermostable and resistant to proteolytic inactivation. The catalytic domains hydrolysed xylan by a typical endo-mode of action. The type VI cellulose-binding domain (CBD) homologue of XylV bound xylan and, to a smaller extent, Avicel and acid-swollen cellulose. Deletion of the CBD from XylV abolished the capacity of the enzymes to bind polysaccharides. The polysaccharide-binding domain was shown to have a key role in the hydrolysis of insoluble substrates by XylV. The C-terminal domain of XylV, which is absent from XylU, removed acetyl groups from acetylated xylan and acted in synergy with the glycosyl hydrolase catalytic domain of the enzyme to elicit the hydrolysis of acetylated xylan.

Acetylation↗

PubMed2001

Degradation of cellulose by nitrogen-fixing strain of Bacillus polymyxa.

During an intensive screening programme, several strains of cellulolytic bacteria were isolated. One nitrogenase-positive strain able to degrade filter paper, Avicel cellulose, carboxymethyl cellulose and cellobiose was selected for further study. On the basis of biochemical characteristics and Mol % of G+C content, the selected strain was identified as Bacillus polymyxa. The highest production of the enzymes degrading filter paper (FP-ase) and carboxymethyl cellulose (CMCase) by B. polymyxa was observed in Park's medium suplemented with Avicel cellulose. The investigated strain of bacteria produced cellulosome-like structures as was shown by transmission electron microscopy.

Bacillus↗

PubMed2003

Application of microbial genes to recalcitrant biomass utilization and environmental conservation.

Recent papers concerning the application of microbial genes to recalcitrant biomass utilization and environmental conservation are reviewed. Microbial genes have been integrated and expressed in plants and microorganisms. When cellulose-degrading enzyme genes are expressed in rice plants, the transgenic plants exhibit swollen cell walls which increases the digestibility of rice straw in the rumen. When genes encoding aromatic compound-degrading enzymes are expressed in plants, it is expected that aromatic compounds contaminating soil would be degraded during the growth of the transgenic plants. The former transgenic plants are utilized as feed and the latter for phytoremediation. Dockerin and cohesin interactions occurring in the cellulase complex, cellulosome, are applied to the construction of artificial enzyme complexes and protein purification by expressing the genes in transformed bacteria and/or silkworms, respectively. In the case of the forced expression of bacterial genes encoding chitinase and/or hydrogenase in the wild-type bacteria, chitin degradation and hydrogen gas production in the transformed bacteria occur at much higher rates than in the wild type.

Journal Article↗

PubMedDate not supplied

[Obtaining of ScFv-CBD fusion protein and its application for affinity purification of recombinant human interferon alpha2b].

The gene of ScFv-CBD-fusion protein has been designed using the DNA sequences encoding of single-chain antibody (ScFv) against human interferon alpha2b (IFN-alpha2b) and cellulose-binding domain (CBD) from Clostridium thermocellum cellulosome. Biosynthesis of ScFv-CBD utilizing high-productive Escherichia coli system was carried out and the accumulation of target protein in bacterial inclusion bodies was shown. After the purification of the inclusion bodies and their subsequent in vitro refolding the soluble ScFv-CBD-fusion protein was directly immobilized on cellulose by bioaffinity coupling. The possibility to obtain the preparative quantities of ScFv-CBD in biologically-active form using different refolding schemes was accurately investigated in the paper. The general applicability of biologically immobilized ScFv-CBD-fusion proteins for affinity purification of recombinant IFN-alpha2b is shown.

Antibodies, Monoclonal↗

PubMed1995

Biomass degrading enzymes from anaerobic rumen fungi.

Obligately anaerobic fungi are part of the natural microflora of the alimentary tract of many herbivorous mammals. They produce a complete set of polysaccharide hydrolytic enzymes which efficiently degrade plant cell-walls. This article summarizes the present work on biomass degrading enzymes with special emphasis on their cellulases, xylanases, and esterases from both monocentric and polycentric anaerobic fungi. The possibility of the cellulosome or xylanosome-like high molecular mass complexes in the fungi are also discussed.

Anaerobiosis↗

PubMed1996

Crystal structure of a bacterial family-III cellulose-binding domain: a general mechanism for attachment to cellulose.

The crystal structure of a family-III cellulose-binding domain (CBD) from the cellulosomal scaffoldin subunit of Clostridium thermocellum has been determined at 1.75 A resolution. The protein forms a nine-stranded beta sandwich with a jelly roll topology and binds a calcium ion. conserved, surface-exposed residues map into two defined surfaces located on opposite sides of the molecule. One of these faces is dominated by a planar linear strip of aromatic and polar residues which are proposed to interact with crystalline cellulose. The other conserved residues are contained in a shallow groove, the function of which is currently unknown, and which has not been observed previously in other families of CBDs. On the basis of modeling studies combined with comparisons of recently determined NMR structures for other CBDs, a general model for the binding of CBDs to cellulose is presented. Although the proposed binding of the CBD to cellulose is essentially a surface interaction, specific types and combinations of amino acids appear to interact selectively with glucose moieties positioned on three adjacent chains of the cellulose surface. The major interaction is characterized by the planar strip of aromatic residues, which align along one of the chains. In addition, polar amino acid residues are proposed to anchor the CBD molecule to two other adjacent chains of crystalline cellulose.

Amino Acid Sequence↗

PubMed2005

Genomic analysis of the protein secretion systems in Clostridium acetobutylicum ATCC 824.

Consistent information about protein secretion in Gram-positive bacteria is essentially restricted to the model organism Bacillus subtilis. Among genome-sequenced clostridia, Clostridium acetobutylicum has been the most extensively studied from a physiological point of view and is the organism for which the largest variety of molecular biology tools have been developed. Following in silico analyses, both secreted proteins and protein secretion systems were identified. The Tat (Twin arginine translocation; TC #2.A.64) pathway and ABC (ATP binding cassette) protein exporters (TC #3.A.1.) could not be identified, but the Sec (secretion) pathway (TC #3.A.5) appears to be used prevalently. Similarly, a flagella export apparatus (FEA; TC #3.A.6.), holins (TC #1.E.), and an ESAT-6/WXG100 (early secreted antigen target of 6 kDa/proteins with a WXG motif of approximately 100 residues) secretion system were identified. Here, we report for the first time the identification of a fimbrilin protein exporter (FPE; TC #3.A.14) and a Tad (tight adherence) export apparatus in C. acetobutylicum. This investigation highlights the potential use of this saprophytic bacterium in biotechnological and biomedical applications as well as a model organism for studying protein secretion in pathogenic Gram-positive bacteria.

Amino Acid Sequence↗

PubMed2006

Protein cell surface display in Gram-positive bacteria: from single protein to macromolecular protein structure.

In the course of evolution, Gram-positive bacteria, defined here as prokaryotes from the domain Bacteria with a cell envelope composed of one biological membrane (monodermita) and a cell wall composed at least of peptidoglycan and covalently linked teichoic acids, have developed several mechanisms permitting to a cytoplasmic synthesized protein to be present on the bacterial cell surface. Four major types of cell surface displayed proteins are currently recognized: (i) transmembrane proteins, (ii) lipoproteins, (iii) LPXTG-like proteins and (iv) cell wall binding proteins. The subset of proteins exposed on the bacterial cell surface, and thus interacting with extracellular milieu, constitutes the surfaceome. Here, we review exhaustively the current molecular mechanisms involved in protein attachment within the cell envelope of Gram-positive bacteria, from single protein to macromolecular protein structure.

Amino Acid Sequence↗