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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.

Background: Bioethanol production processes involve enzymatic hydrolysis of pretreated lignocellulosic biomass into fermentable sugars. Due to the relatively high cost of enzyme production, the development of potent and costeffective cellulolytic cocktails is critical for increasing the cost-effectiveness of bioethanol production. In this context, the multi-protein cellulolytic complex of Clostridium (Ruminiclostridium) thermocellum, the cellulosome, was studied here. C. thermocellum is known to assemble cellulosomes of various subunit (enzyme) compositions, in response to the available carbon source. In the current study, different carbon sources were used, and their influence on both cellulosomal composition and the resultant activity was investigated. Results: Glucose, cellobiose, microcrystalline cellulose, alkaline-pretreated switchgrass, alkaline-pretreated corn stover, and dilute acid-pretreated corn stover were used as sole carbon sources in the growth media of C. thermocellum strain DSM 1313. The purified cellulosomes were compared for their activity on selected cellulosic substrates. Interestingly, cellulosomes derived from cells grown on lignocellulosic biomass showed no advantage in hydrolyzing the original carbon source used for their production. Instead, microcrystalline cellulose- and glucose-derived cellulosomes were equal or superior in their capacity to deconstruct lignocellulosic biomass. Mass spectrometry analysis revealed differential composition of catalytic and structural subunits (scaffoldins) in the different cellulosome samples. The most abundant catalytic subunits in all cellulosome types include Cel48S, Cel9K, Cel9Q, Cel9R, and Cel5G. Microcrystalline cellulose- and glucose-derived cellulosome samples showed higher endoglucanase-to-exoglucanase ratios and higher catalytic subunit-per-scaffoldin ratios compared to lignocellulose-derived cellulosome types. Conclusion: The results reported here highlight the finding that cellulosomes derived from cells grown on glucose and microcrystalline cellulose are more efficient in their action on cellulosic substrates than other cellulosome preparations. These results should be considered in the future development of C. thermocellum-based cellulolytic cocktails, designer cellulosomes, or engineering of improved strains for deconstruction of lignocellulosic biomass. [ABSTRACT FROM AUTHOR]

Background: Expansins are relatively small proteins that lack enzymatic activity and are found in plants and microorganisms. The function of these proteins is to disrupt the plant cell walls by interfering with the non-covalent interchain bonding of the polysaccharides. Expansins were found to be important for plant growth, but they are also expressed by various bacteria known to have interactions with plants. Clostridium clariflavum is a plant cell walldegrading bacterium with a highly elaborate cellulosomal system. Among its numerous dockerin-containing genes, two expansin-like proteins, Clocl_1862 and Clocl_1298 (termed herein CclEXL1 and CclEXL2) were identified, and CclEXL1 was found to be expressed as part of the cellulosome system. This is the first time that an expansin-like protein is identified in a cellulosome complex, which implicates its possible role in biomass deconstruction. Results: In the present article, we analyzed the functionality of CclEXL1. Its dockerin was characterized and shown to bind selectively to type-I cohesins of C. clariflavum, with preferential binding to the cohesin of ScaG, and additionally to a type-I cohesin of C. cellulolyticum. We demonstrated experimentally that the expansin-like protein binds preferentially to microcrystalline cellulose, but it also binds to acid-swollen cellulose, xylan, and wheat straw. CclEXL1 exhibited a pronounced loosening effect on filter paper, which resulted in substantial decrease in tensile stress. The C. clariflavum expansin-like protein thus enhances significantly enzymatic hydrolysis of cellulose, both by C. clariflavum cellulosomes and two major cellulosomal cellulases from this bacterium: GH48 (exoglucanase) and GH9 (endoglucanase). Finally, we demonstrated CclEXL1-mediated enhancement of microcrystalline cellulose degradation by different cellulosome fractions and the two enzymes. Conclusions: The results of this study confirm that the C. clariflavum expansin-like protein is part of the elaborate cellulosome system of this bacterium with capabilities of cellulose creeping. The data suggest that pretreatment of cellulosic materials with CclEXL1 can bring about substantial improvement of hydrolysis by cellulases. [ABSTRACT FROM AUTHOR]

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]

Cellular behaviors and a xylanolytic-cellulolytic enzyme system of Tepidimicrobium xylanilyticum BT14 towards xylan-rich plant biomass degradation were characterized. During the exponential growth phase, the bacterial cells were bound tightly to the growth substrate where the degradation zones appeared mostly around the cells, indicating that the xylanolytic-cellulolytic enzyme system was linked to the cell surfaces. Interestingly, several cells appeared to secrete extracellular matrix to connect to their neighbors, and the matrix disappeared when cells passed to the stationary growth phase. Cationized-ferritin staining resulted in a dense assembly of bulbs, protuberance-like structures on the growing bacterial cell surfaces. The cell-associated proteins derived by sonication contained predominated xylanase with relatively low carboxymethyl-cellulase (CMCase) activities, suggesting that the xylanolytic-cellulolytic enzyme system occurred as a cell-associated enzyme. By means of gel-filtration chromatography, a high molecular mass protein with the estimated size of 2000 kDa was retrieved from the cell-associated enzymes, and it appeared as a single protein band on non-denaturing gel. However, more bands were obtained after the protein was boiled with sodium dodecyl sulfate and β-mercaptoethanol - which contained 4 xylanases and one CMCase - suggesting that these proteins were organized as a multienzyme complex (MEC) in natural form. Additionally, the predominated xylanolytic MEC preferred binding to cellulose rather than xylan. [ABSTRACT FROM AUTHOR]

The effect of biomass loading from 50 to 200 g/L on enzymatic hydrolysis was studied using switchgrass samples pretreated by dilute acid and hypochlorite-alkaline methods. It was confirmed that an increase of initial loading of the pretreated biomass leads to a decrease of enzymatic digestibility, probably due to difficulty of mass transfer of cellulolytic enzymes in the high-viscous substrate slurry and also because of an inhibiting effect of the formed sugars. An additional sharp problem connected with enzymatic hydrolysis at the high-solids loading is absorption and retention of liquid hydrolysate by residual non-hydrolyzed biomass that causes diminution of the available volume (Va) of the sugar solution and decreases productivity of the saccharification process. To optimize the high-solids enzymatic hydrolysis, the maximal amount of the formed sugars was determined Am = Cm x Va,m , where Cm is maximal concentration of the sugar solution and Va,m is maximal available volume. Such an approach makes it possible to find the optimal conditions for the hydrolysis: optimal biomass loading and hydrolysis time. After enzymatic hydrolysis at these optimal conditions, the low-lignified biomass pretreated by hypochlorite-alkaline method displayed much more available volume of sugar solution and higher digestibility characteristics than the cellolignin obtained by acidic pretreatment of the initial biomass sample. [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 effect of mild acidic and alkaline pretreatments of various plant biomasses on their enzymatic hydrolysis has been studied. The yield of reducing sugars and utilization rate of the biomass were used as reliable characteristics of enzymatic digestibility of the biomasses. The experiments showed that alkaline pretreatment was more efficient than acidic pretreatment. As a result of alkaline pretreatment, a more efficient delignification of the biomasses and considerable improvement of the digestibility parameters were observed. It was found that residual lignin content in the biomass after alkaline pretreatment was related to initial lignin content in untreated biomass. Moreover, residual lignin showed an evident negative effect on enzymatic hydrolysis of pretreated biomass samples, and its removal contributed to higher enzymatic digestibility. It is more preferred to select a mild alkaline pretreatment for biomass that has low content of initial lignin. Such treatment yielded highly delignified biomass with increased percentage of cellulose fraction, which enhanced digestibility at low enzyme loading with a relatively short hydrolysis time. [ABSTRACT FROM AUTHOR]

Enzymatic hydrolysis of non-dried and dried cellulose samples having various particles size, degree of polymerization, porosity, crystalline polymorph, and content of non-crystalline domains has been studied. Regression analysis was carried out to determine contribution of various structural features of cellulose samples to their hydrolysability. It was found that particle size, degree of polymerization, and crystalline polymorph had a negligible influence on the conversion degree of cellulose into glucose under the effect of the cellulolytic enzyme. Such characteristics as the pores volume had a fair impact on the conversion degree of cellulose. Drying of the wet samples caused decreasing of the hydrolysability of cellulose due to irreversible collapse of the pores volume. The content of non-crystalline domains (Ax) in cellulose had the highest effect on the rate of enzymatic hydrolysis and average conversion degree (aa) of cellulose into glucose. A linear dependence aa = f(Ax) was established both for dried and non-dried cellulose samples. [ABSTRACT FROM AUTHOR]

Efficient degradation of cellulose by the anaerobic thermophilic bacterium, Clostridium thermocellum, is carried out by the multi-enzyme cellulosome complex. The enzymes on the complex are attached in a calcium-dependent manner via their dockerin (Doc) module to a cohesin (Coh) module of the cellulosomal scaffoldin subunit. In this study, we have optimized the Coh-Doc interaction for the purpose of protein affinity purification. A C. thermocellum Coh module was thus fused to a carbohydrate-binding module, and the resultant fusion protein was applied directly onto beaded cellulose, thereby serving as a non-covalent 'activation' procedure. A complementary Doc module was then fused to a model protein target: xylanase T-6 from Geobacillus stearothermophilus. However, the binding to the immobilized Coh was only partially reversible upon treatment with EDTA, and only negligible amounts of the target protein were eluted from the affinity column. In order to improve protein elution, a series of truncated Docs were designed in which the calcium-coordinating function was impaired without appreciably affecting high-affinity binding to Coh. A shortened Doc of only 48 residues was sufficient to function as an effective affinity tag, and highly purified target protein was achieved directly from crude cell extracts in a single step with near-quantitative recovery of the target protein. Effective EDTA-mediated elution of the sequestered protein from the column was the key step of the procedure. The affinity column was reusable and maintained very high levels of capacity upon repeated rounds of loading and elution. Reusable Coh-Doc affinity columns thus provide an efficient and attractive approach for purifying proteins in high yield by modifying the calcium-binding loop of the Doc module. Copyright © 2008 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

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 S (CelS) from the cellulosome of ATCC strain 27405. This component is not related to any other cellulosomal subunit or cloned endoglucanase in this organism. [ABSTRACT FROM AUTHOR]

Clostridium thermocellum, an anaerobic thermophilic cellulolytic bacterium, produces an extremely cohesive, very high-molecular-mass multicellulase-containing complex termed the cellulosome. One of its components, the S1 subunit, is a nonenzymatic, 210 kDa glycopolypeptide. Upon preconditioning of the intact cellulosome with lowionic-strength or low-pH solutions, the S1 subunit separates in hot sodium dodecyl sulfate (SDS) solutions into a series of defined lowermolecular-mass subcomponents. Under the same conditions, the purified S1 subunit demonstrated the same behavior. Higher levels of glycosylation associated with the larger S1 subcomponents. The data support alterations in the conformational state of the S1 structure that lead to its disintegration induced by combined treatments with SDS and heating. Evidence is provided that this phenomenon may reflect a physiological response of the cellulosome, since similar alterations in S1 appear to accompany its binding to cellulose. [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]

β-Glucosidases are key enzymes in the process of cellulose utilization. It is the last enzyme in the cellulose hydrolysis chain, which converts cellobiose to glucose. Since cellobiose is known to have a feedback inhibitory effect on a variety of cellulases, β-glucosidase can prevent this inhibition by hydrolyzing cellobiose to non-inhibitory glucose. While the optimal temperature of the Clostridium thermocellum cellulosome is 70 °C, C. thermocellum β-glucosidase A is almost inactive at such high temperatures. Thus, in the current study, a random mutagenesis directed evolutionary approach was conducted to produce a thermostable mutant with Kcat and Km, similar to those of the wild-type enzyme. The resultant mutant contained two mutations, A17S and K268N, but only the former was found to affect thermostability, whereby the inflection temperature (Ti) was increased by 6.4 °C. A17 is located near the central cavity of the native enzyme. Interestingly, multiple alignments revealed that position 17 is relatively conserved, whereby alanine is replaced only by serine. Upon the addition of the thermostable mutant to the C. thermocellum secretome for subsequent hydrolysis of microcrystalline cellulose at 70 °C, a higher soluble glucose yield (243%) was obtained compared to the activity of the secretome supplemented with the wild-type enzyme. [ABSTRACT FROM AUTHOR]

Abstract: Passive vaccination is used to treat a wide range of infections and cancer. However, this approach has some limitations. An immune complex termed Y-complex was developed to intensify the effect of the passive vaccine. The complex is composed of a microbead that carries specific antibodies and an inducer. It enables targeting of pathogen or abnormal cells, and stimulation of a desired response by innate immune cells, depending on the inducer. The production and efficacy of Y-complex as a passive immune prophylaxis is demonstrated in this study by its use in treating cow mastitis. In an in vitro assay, Y-complex inhibited propagation and induced phagocytosis of bacteria. In challenge experiments, cows were inoculated through the udder with Escherichia coli or Streptococcus dysgalactiae. Following treatment with Y-complex, no bacteria were isolated in the milk and N-acetyl-ß-d-glucosaminidase activity had returned to normal levels. Thus the Y-complex approach can be used as an effective treatment for mastitis. Due to its modularity, this approach may serve as a treatment for a variety of disease agents. [Copyright &y& Elsevier]

The high affinity cohesin-dockerin interaction dictates the suprastructural assembly of the multienzyme cellulosome complex. The connection between affinity and species specificity was studied by exploring the recognition properties of two structurally related cohesin species of divergent specificity. The cohesins were examined by progressive rounds of swapping, in which corresponding homologous stretches were interchanged. The specificity of binding of the resultant chimeric cohesins was determined by enzyme-linked affinity assay and complementary protein microarray. In succeeding rounds, swapped segments were systematically contracted, according to the binding behavior of previously generated chimeras. In the fourth and final round we discerned three residues, reputedly involved in interspecies binding specificity. By replacing only these three residues, we were able to convert the specificity of the resultant mutated cohesin, which bound preferentially to the rival dockerin with ∼20% capacity of the wild-type interaction. These residues represent but 3 of the 16 contact residues that participate in the cohesin-dockerin interaction. This approach allowed us to differentiate, in a structure-independent fashion, between residues critical for interspecies recognition and binding residues per se. [ABSTRACT FROM AUTHOR]