Biomedical subjects

E Morag

Publications and source records attributed to E Morag.

29 records · Page 2Linked to original sources

PubMed1995

Expression, purification, and characterization of the cellulose-binding domain of the scaffoldin subunit from the cellulosome of Clostridium thermocellum.

The major cellulose-binding domain (CBD) from the cellulosome of Clostridium thermocellum YS was cloned and overexpressed in Escherichia coli. The expressed protein was purified efficiently by a modification of a novel procedure termed affinity digestion. The properties of the purified polypeptide were compared with those of a related CBD derived from a cellulosome-like complex of a similar (but mesophilic) clostridial species, Clostridium cellulovorans. The binding properties of the two proteins with their common substrate were found to be very similar. Despite the similarity in the amino acid sequences of the two CBDs, polyclonal antibodies raised against the CBD from C. thermocellum failed to interact with the protein from C. cellulovorans. Chemical modification of the single cysteine of the CBD had little effect on the binding to cellulose. Biotinylation of this cysteine allowed the efficient binding of avidin to cellulose, and the resultant matrix is appropriate for use as a universal affinity system.

Affinity Labels↗

PubMed1994

Crystallization and preliminary X-ray analysis of the major cellulose-binding domain of the cellulosome from Clostridium thermocellum.

The cellulose-binding domain from the scaffoldin subunit of the cellulosome from Clostridium thermocellum strain YS has been expressed in Escherichia coli, purified to homogeneity, and crystallized. Crystals were grown by vapor diffusion using polyethylene glycol as precipitant. They belong to the monoclinic space group C2 with unit cell dimensions of a = 64.68 A, b = 50.36 A, c = 96.27 A; beta = 99.43 degrees, and density packing considerations suggest that the asymmetric unit contains two molecules. The crystals diffract beyond 2.0 A resolution using a laboratory rotating anode source.

Cellulase↗

PubMed1994

The cellulosome--a treasure-trove for biotechnology.

The cellulases of many cellulolytic bacteria are organized into discrete multienzyme complexes, called cellulosomes. The multiple subunits of cellulosomes are composed of numerous functional domains, which interact with each other and with the cellulosic substrate. One of these subunits comprises a distinctive new class of noncatalytic scaffolding polypeptide, which selectively integrates the various cellulase and xylanase subunits into the cohesive complex. Intelligent application of cellulosome hybrids and chimeric constructs of cellulosomal domains should enable better use of cellulosic biomass and may offer a wide range of novel applications in research, medicine and industry.

Bacteria↗

PubMed1993

The nature of the carbohydrate-peptide linkage region in glycoproteins from the cellulosomes of Clostridium thermocellum and Bacteroides cellulosolvens.

The cellulase complexes of two cellulolytic bacteria, Clostridium thermocellum and Bacteroides cellulosolvens, were subjected to extensive Pronase digestion. Glycopeptide fractions were isolated by gel permeation and fast protein liquid chromatography and analyzed by monosaccharide analysis, amino acid analysis, methylation analysis, and 1H NMR spectroscopy. Alkaline borohydride-induced deglycosylation/amino acid conversion and periodate oxidation studies on the glycopeptide fraction of the C. thermocellum cellulosome demonstrated that the earlier established collection of carbohydrate moieties with 3-O-Me-D-GlcpNAc-alpha (1-->2)-[D-Galp-alpha (1-->3)]-D-Galf-alpha (1-->2)-D-Gal (where 3-O-Me-D-GlcpNAc is 3-O-methyl-N-acetylglucopyranosamine, Galp is galactopyranose, and Galf is galactofuranose) as the major component, is O-linked to threonine via galactopyranose. Using the same approach for the glycopeptide fraction of the cellulase complex of B. cellulosolvens, it was found that the reported collection of carbohydrate moieties with D-Galf-alpha (1-->3)-D-GlcpNAc-alpha (1-->2)-D-Galf-alpha (1-->2)-[D-Galf-beta (1-->3)]-D-Gal as the major component, is O-linked mainly to threonine and partly to serine via galactopyranose. In both species, the hydroxyamino-acid-bound galactopyranose residue has probably an alpha-configuration. The carbohydrate chains appear as clusters located in highly Thr/Pro-rich peptide regions of the glycoproteins. The results are consistent with the notion that the glycosylation sites are localized in linker sequences which connect the various binding domains of the noncatalytic S1 subunit of the cellulosome.

Bacteroides↗

PubMed1993

Cellulase Ss (CelS) is synonymous with the major cellobiohydrolase (subunit S8) from the cellulosome of Clostridium thermocellum.

The controversy regarding the identity of a major cellulosomal component type from two different strains of Clostridium thermocellum has been resolved. The principal cellobiohydrolase, subunit S8, from the cellulosome of strain YS has been demonstrated to be synonymous with cellulase component Ss (CelS) from the cellulosome of ATCC strain 27405. This component is not related to any other cellulosomal subunit or cloned endoglucanase in this organism.

Amino Acid Sequence↗

PubMed1992

Identification of the cellulose-binding domain of the cellulosome subunit S1 from Clostridium thermocellum YS.

The 3' region of a gene designated cipB, which shows strong homology with cipA that encodes the cellulosome SL subunit of Clostridium thermocellum ATCC 27405, was isolated from a gene library of C. thermocellum strain YS. The truncated S1 protein encoded by the cipB derivative bound tightly to cellulose. The cellulose-binding domain in this polypeptide consisted of a C-terminal proximal 167 residue sequence which showed complete identity with residues 337-503 of mature SL from C. thermocellum strain ATCC 27405. The cellulose-binding domain interacted with both crystalline and amorphous cellulose, but not with xylan.

Amino Acid Sequence↗

PubMed1992

Novel oligosaccharide constituents of the cellulase complex of Bacteroides cellulosolvens.

The multiple cellulase-containing protein complex, isolated from the cellulolytic bacterium Bacteroides cellulosolvens, contains oligosaccharides which are O-linked mainly to a 230-kDa subunit. The oligosaccharide chains were liberated by alkaline-borohydride treatment and fractionated as oligosaccharide alditols via gel-permeation chromatography and HPLC. The fractions were investigated by one- and two-dimensional (correlation, homonuclear Hartmann-Hahn, rotating-frame nuclear Overhauser enhancement) 500-MHz 1H-NMR spectroscopy in combination with monosaccharide and methylation analyses and with fast-atom-bombardment mass spectrometry. The following carbohydrate structures could be established: [formula: see text] The results indicate an interesting similarity between the oligosaccharide moieties of the cellulase complex of B. cellulosolvens and of Clostridium thermocellum [Gerwig, G. J., Kamerling, J. P., Vliegenthart, J. F. G., Morag (Morgenstern), E., Lamed, R. & Bayer, E. A. (1991) Eur. J. Biochem. 196, 115-122], having 3, 5 and 6 as common elements. The furanose form of a terminal alpha-D-galactose residue demonstrated an inhibitory effect on the interaction of Griffonia simplicifolia I isolectin B4 with the cellulosome-like entity of B. cellulosolvens.

Amidohydrolases↗

PubMed1991

Primary structure of O-linked carbohydrate chains in the cellulosome of different Clostridium thermocellum strains.

The cell-free forms of the multiple cellulase-containing protein complex (cellulosome), isolated from the cellulolytic bacterium Clostridium thermocellum strains YS, ATCC 27405 and LQRI, have a total carbohydrate content of 5-7% (by mass), consisting of O-linked oligosaccharide chains. The carbohydrate chains were liberated by alkaline-borohydride treatment and fractionated as oligosaccharide alditols via gel-permeation chromatography and HPLC. The fractions were investigated by 500-MHz 1H-NMR spectroscopy in combination with monosaccharide and methylation analysis and with fast-atom-bombardment mass spectrometry (FAB-MS). In addition to the previously described major oligosaccharide, (formula; see text) [Gerwig, G. J., de Waard, P., Kamerling, J. P., Vliegenthart, J. F. G., Morgenstern, E., Lamed, R. & Bayer, E. A. (1989) J. Biol. Chem. 264, 1027-1035], the following partial structures of this compound could be established: (formula; see text). Cell-free and cell-associated forms of the cellulosome of C. thermocellum, as determined for strain YS, have the same oligosaccharide pattern. Based on the oligosaccharide structures, a biosynthetic pathway is suggested.

Bacterial Proteins↗

PubMed1991

Anomalous dissociative behavior of the major glycosylated component of the cellulosome of Clostridium thermocellum.

The cellulosome of Clostridium thermocellum is a highly cohesive multienzyme complex that is capable of completely solubilizing insoluble cellulose. One of the major cellulosomal components, the glycosylated S1 subunit, is believed to play an important structural role and normally migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an Mr of 210,000. It is shown here that by simply altering the conditions (pH or ionic strength) of the environment prior to electrophoresis, a different migratory profile for S1 emerges, yielding a collection of bands, all of which migrate faster than the parent band. The original electrophoretic behavior of S1 can be reproduced on restoration of the original pH and ionic strength. These results may bear important significance for the physiological role of the S1 subunit in facilitating the observed synergistic action of the other (cellulolytic) components of the cellulosome.

Biotechnology↗

PubMed1991

Isolation and properties of a major cellobiohydrolase from the cellulosome of Clostridium thermocellum.

In the anaerobic, thermophilic, cellulolytic bacterium Clostridium thermocellum, efficient solubilization of the insoluble cellulose substrate is accomplished largely through the action of a cellulose-binding multienzyme complex, the cellulosome. A major cellobiohydrolase activity from the cellulosome has been traced to its Mr 75,000 S8 subunit, and an active fragment of this subunit was prepared by a novel procedure involving limited proteolytic cleavage. The truncated Mr 68,000 fragment, termed S8-tr, was purified by gel filtration and high-performance ion-exchange chromatography. The purified protein adsorbed weakly to amorphous cellulose, and its enzymatic action yielded cellobiose as the major end product from both amorphous and crystalline cellulose preparations. The high ratio of exo- to endo-beta-glucanase activities was supported by viscosimetric measurements. The use of model substrates showed that the smallest cellodextrin to be degraded was cellotetraose, but cellopentaose was degraded at a much greater rate. Cellobiose dramatically inhibited the cellulolytic activities. In the absence of calcium or other bivalent metal ions, both the truncated cellobiohydrolase activity of S8-tr and the true cellulase activity of the parent cellulosome were relatively unstable at temperatures above 50 degrees C. Cysteine further enhanced the stabilizing effect of calcium. This is the first report of a defined cellobiohydrolase in C. thermocellum. Its association with the cellulosome and the correspondence of several of their major distinctive properties suggest that this cellobiohydrolase plays a key role in the solubilization of cellulose by the intact cellulosomal complex.

Animals↗

PubMed1990

Relationship of cellulosomal and noncellulosomal xylanases of Clostridium thermocellum to cellulose-degrading enzymes.

Xylanase activity of Clostridium thermocellum, an anaerobic thermophilic cellulolytic bacterium, was characterized. The activity was localized both in the cellulosome (the principal multienzyme, cellulose-solubilizing protein complex) and in noncellulosomal fractions. Each of these fractions contained at least four major polypeptide bands which contributed to the xylanolytic activity. In both cases, pH and temperature optima, product pattern, and other features of the xylanase activity were almost identical. The main difference was in the average molecular weights of the respective polypeptides which appeared responsible for the activity. In the noncellulosomal fraction, xylanases with Mrs ranging from 30,000 to 65,000 were detected. Distinct from these were the cellulosomal xylanases, which exhibited much larger Mrs (up to 170,000). The cellulosome-associated xylanases corresponded to known cellulosomal subunits, some of which also exhibited endoglucanase activity, and others which coincided with subunits which appeared to express exoglucanaselike activity. In contrast, the noncellulosomal xylanases hydrolyzed xylan exclusively. beta-Glucosidase and beta-xylosidase activities were shown to be the action of different enzymes; both were associated exclusively with the cell and were not components of the cellulosome. Despite the lack of growth on and utilization of xylan or its degradation products, C. thermocellum produces a highly developed xylanolytic apparatus which is interlinked with its cellulase system.

Bacterial Adhesion↗