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The cellulose-binding domain (CBD) present on a noncatalytic scaffoldin subunit of the cellulosome in Clostridium thermocellum is similar in structure, substrate affinity and binding capacity to the CBD present in C. cellulovorans. However the polyclonal antibodies produced against the CBD of C. thermocellum do not act against the CBD of C. cellulovorans possibly because the exposed epitopes in the two CBD's are different. Chemical changes in the cysteine of the CBD do not affect the CBD's ability to bind cellulose.

Results show that the cellulosomal gene celS is regulated by growth rate in Clostridium thermocellum. The gene encoded product CelS exhibits exoglucanase activity. Data indicae that the amount of CelS produced by cellobiose-grown cultures is lower than that of cellulose-grown cultures.

The cellulosome of the cellulolytic bacterium Clostridium thermocellum has a structural multi-modular protein called CipA (cellulosome-integrating protein A) that includes nine enzyme-binding cohesin modules and a family 3 cellulose-binding module (CBM3a). In the CipA protein, the CBM3a module is located between the second and third cohesin modules and is connected to them via proline/threonine-rich linkers. The structure of CBM3a with portions of the C- and N-terminal flanking linker regions, CBM3a-L, has been determined to a resolution of 1.98 Å. The structure is a β-sandwich with a structural Ca2+ ion. The structure is consistent with the previously determined CipA CBM structure; however, the structured linker regions provide a deeper insight into the overall cellulosome structure and assembly. [ABSTRACT FROM AUTHOR]

The conversion of recalcitrant plant-derived cellulosic biomass into biofuels is dependent on highly efficient cellulase systems that produce near-quantitative levels of soluble saccharides. Similar to other fungal and bacterial cellulase systems, the multienzyme cellulosome system of the anaerobic, cellulolytic bacterium Clostridium thermocellum is strongly inhibited by the major end product cellobiose. Cellobiose-induced inhibition can be relieved via its cleavage to noninhibitory glucose by the addition of exogenous noncellulosomal enzyme ß-glucosidase; however, because the cellulosome is adsorbed to the insoluble substrate only a fraction of ß-glucosidase would be available to the cellulosome. Towards this end, we designed a chimeric cohesin-fused ß-glucosidase (BglACohll) that binds directly to the cellulosome through an unoccupied dockerin module of its major scaffoldin subunit. The &bgr;-glucosidase activity is thus focused at the immediate site of cellobiose production by the cellulosomal enzymes. BglA-Cohll was shown to retain cellobiase activity and was readily incorporated into the native cellulosome complex. Surprisingly, it was found that the native C. thermocellum cellulosome exists as a homooligomer and the high-affinity interaction of BglA-Cohll with the scaffoldin moiety appears to dissociate the oligomeric state of the cellulosome. Complexation of the cellulosome and BglA-Cohll resulted in higher overall degradation of microcrystalline cellulose and pretreated switchgrass compared to the native cellulosome alone or in combination with wild-type BgIA in solution. These results demonstrate the effect of enzyme targeting and its potential for enhanced degradation of cellulosic biomass. [ABSTRACT FROM AUTHOR]

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, rotatingframe nuclear Overhauser enhancement) 500-MHz ¹H-NMR spectroscopy in combination with monosaccharide and methylation analyses and with fast-atom-bombardment mass spectrometry. 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 α-D-galactose residue demonstrated an inhibitory effect on the interaction of Griffonia simplicifolia I isolectin B4 with the cellulosome-like entity of B. cellulosolvens. [ABSTRACT FROM AUTHOR]

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, Multiple line equations cannot be represented in ASCII 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: Multiple line equations cannot be represented in ASCII text. I 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. [ABSTRACT FROM AUTHOR]