Thermocellum Cellulase Research Articles

A novel Ruminiclostridium thermocellum cellulase system enhances cellulosic saccharification by elimination of cellobiose feedback inhibition

The Ruminiclostridium thermocellum cellulosome system is one of the most efficient cellulase systems in nature. The main hydrolysis product of cellulosic saccharification by R. thermocellum was cellobiose, which served as a strong feedback inhibitor to the cellulosome. In this study, R. thermocellum M3, which directly hydrolyzes cellulosic biomass into monosaccharides, was used to degrade cellobiose to investigate the kinetic characteristics of cellobiose degradation. Moreover, the transcriptomic characteristics of strain M3 under cellobiose and Avicel culture conditions were compared. The results indicated that strain M3 was able to grow and hydrolyze cellobiose under cellobiose concentrations higher than 20 g/L. The best growth performance and glucose production were obtained under 12 g/L cellobiose. Compared with that of Avicel, cellobiose had greater β-glucosidase activity under cellobiose culture, with a maximum glucose yield of 767.97±19.13 mg/g cellobiose. The transcription results indicated that R. thermocellum M3 resisted the feedback inhibition of cellobiose by regulating genes encoding β-glucosidase, cellobiose phosphorylase and ABC transporters. The high glucose yield of strain M3 was closely related to the expression of the bglX gene. In addition, the gene related to the chemotactic pathway of strain M3 also increased its perception and adaptation to cellobiose.

Stable cellulolytic activity of Clostridium thermocellum against cellulosic biomass pretreated with ionic liquid 1-ethyl 3-methylimidazolium acetate

Using cellulosic biomass has considerable potential for the production of valuable chemicals while contributing to carbon neutrality. This study investigates the effect of biomass pretreatment with ionic liquid 1-ethyl 3-methylimidazolium acetate ([EMIM][Ac]) on the efficiency of enzymatic saccharification using the cellulolytic system of the thermophilic bacterium Clostridium thermocellum. Although [EMIM][Ac] pretreatment improves the efficiency of enzymatic saccharification, it also inhibits the cellulolytic enzyme. Following incubation with 30 % [EMIM][Ac], while the cellulolytic activity of the commercial cellulase cocktail (Celluclast) decreased to 15 % and that of C. thermocellum exoproteome remained stable, suggesting a relative tolerance of C. thermocellum cellulases to [EMIM][Ac]. The reducing sugars released from the pretreated cellulosic biomass corn hull by Celluclast were reduced by 24 % with supplementation of 5 % [EMIM][Ac] but remained unchanged for C. thermocellum exoproteome. These results highlight the potential of [EMIM][Ac] pretreatment of cellulosic biomass as a promising approach for enzymatic saccharification using C. thermocellum.

Coculture with hemicellulose-fermenting microbes reverses inhibition of corn fiber solubilization by Clostridium thermocellum at elevated solids loadings

BackgroundThe cellulolytic thermophile Clostridium thermocellum is an important biocatalyst due to its ability to solubilize lignocellulosic feedstocks without the need for pretreatment or exogenous enzyme addition. At low concentrations of substrate, C. thermocellum can solubilize corn fiber > 95% in 5 days, but solubilization declines markedly at substrate concentrations higher than 20 g/L. This differs for model cellulose like Avicel, on which the maximum solubilization rate increases in proportion to substrate concentration. The goal of this study was to examine fermentation at increasing corn fiber concentrations and investigate possible reasons for declining performance.ResultsThe rate of growth of C. thermocellum on corn fiber, inferred from CipA scaffoldin levels measured by LC–MS/MS, showed very little increase with increasing solids loading. To test for inhibition, we evaluated the effects of spent broth on growth and cellulase activity. The liquids remaining after corn fiber fermentation were found to be strongly inhibitory to growth on cellobiose, a substrate that does not require cellulose hydrolysis. Additionally, the hydrolytic activity of C. thermocellum cellulase was also reduced to less-than half by adding spent broth. Noting that > 15 g/L hemicellulose oligosaccharides accumulated in the spent broth of a 40 g/L corn fiber fermentation, we tested the effect of various model carbohydrates on growth on cellobiose and Avicel. Some compounds like xylooligosaccharides caused a decline in cellulolytic activity and a reduction in the maximum solubilization rate on Avicel. However, there were no relevant model compounds that could replicate the strong inhibition by spent broth on C. thermocellum growth on cellobiose. Cocultures of C. thermocellum with hemicellulose-consuming partners—Herbinix spp. strain LL1355 and Thermoanaerobacterium thermosaccharolyticum—exhibited lower levels of unfermented hemicellulose hydrolysis products, a doubling of the maximum solubilization rate, and final solubilization increased from 67 to 93%.ConclusionsThis study documents inhibition of C. thermocellum with increasing corn fiber concentration and demonstrates inhibition of cellulase activity by xylooligosaccharides, but further work is needed to understand why growth on cellobiose was inhibited by corn fiber fermentation broth. Our results support the importance of hemicellulose-utilizing coculture partners to augment C. thermocellum in the fermentation of lignocellulosic feedstocks at high solids loading.

Characterization of reduced carbohydrate solubilization during Clostridium thermocellum fermentation with high switchgrass concentrations

Biological conversion of lignocellulosic biomass requires high solids concentration for economical recovery of end products. Clostridium thermocellum fermentations were conducted at various concentrations of switchgrass. Significant reduction of carbohydrate solubilization has been observed for switchgrass with increasing solids concentration. Tests were conducted to understand the cause of inhibition at high solids concentration. Avicel hydrolysis by cell-free C. thermocellum cellulase or C. thermocellum fermentation on cellobiose, supplemented with supernatant or solids from C. thermocellum culture on switchgrass, revealed that inhibition was from the supernatant but not from the solids. Culture of C. thermocellum on Avicel, supplemented with supernatant from C. thermocellum monoculture or mixed consortia, indicated that Avicel solubilization was reduced by almost two-fold with the monoculture supernatant while no significant reduction was observed for the mixed consortia supernatant. Adding 8 kg/m3 commercial xylooligomers during C. thermocellum culture on Avicel reduced Avicel consumption up to 90%. Simultaneous consumption of xylooligomers was suggested for industrial application of C. thermocellum culture on high solids concentration.

Evaluation of promoter sequences for the secretory production of a Clostridium thermocellum cellulase in Paenibacillus polymyxa.

Due to their high secretion capacity, Gram-positive bacteria from the genus Bacillus are important expression hosts for the high-yield production of enzymes in industrial biotechnology; however, to date, strains from only few Bacillus species are used for enzyme production at industrial scale. Herein, we introduce Paenibacillus polymyxa DSM 292, a member of a different genus, as a novel host for secretory protein production. The model gene cel8A from Clostridium thermocellum was chosen as an easily detectable reporter gene with industrial relevance to demonstrate heterologous expression and secretion in P. polymyxa. The yield of the secreted cellulase Cel8A protein was increased by optimizing the expression medium and testing several promoter sequences in the expression plasmid pBACOV. Quantitative mass spectrometry was used to analyze the secretome in order to identify promising new promoter sequences from the P. polymyxa genome itself. The most abundantly secreted host proteins were identified, and the promoters regulating the expression of their corresponding genes were selected. Eleven promoter sequences were cloned and tested, including well-characterized promoters from Bacillus subtilis and Bacillus megaterium. The best result was achieved with the promoter for the hypothetical protein PPOLYM_03468 from P. polymyxa. In combination with the optimized expression medium, this promoter enabled the production of 5475U/l of Cel8A, which represents a 6.2-fold increase compared to the reference promoter PaprE. The set of promoters described in this work covers a broad range of promoter strengths useful for heterologous expression in the new host P. polymyxa.

Hydrolysis of Cellulose by Soluble Clostridium Thermocellum and Acidothermus Cellulolyticus Cellulases

The goal of this work was to clone, express, characterize and assemble a set of soluble thermostablecellulases capable of significantly degrading cellulose. We successfully cloned, expressed, and purified eleven Clostridium thermocellum (Cthe) cellulases and eight Acidothermuscellulolyticus(Acel) cellulases. The performance of the nineteen enzymes was evaluated on crystalline (filter paper) and amorphous (PASC) cellulose. Hydrolysis products generated from these two substrates were converted to glucose using beta-glucosidase and the glucose formed was determined enzymatically. Ten of the eleven Cthe enzymes were highly active on amorphous cellulose. The individual enzymes all produced <10% reducing sugar equivalents from filter paper. Combinations of Cthe cellulases gave higher conversions, with the combination of CelE, CelI, CelG, and CelK converting 34% of the crystalline cellulose. All eight Acel cellulases showed endo-cellulase activity and were highly active on PASC. Only Acel_0615 produced more than 10% reducing sugar equivalents from filter paper, and a combination of six Acel cellulases produced 32% conversion. Acel_0617, a GH48 exo-cellulase, and Acel_0619, a GH12 endo-cellulase, synergistically stimulated cellulose degradation by the combination of Cthe cellulases to almost 80%. Addition of both Acel enzymes to the Cthe enzyme mix did not further stimulate hydrolysis. Cthe CelG and CelI stimulated cellulose degradation by the combination of Acel cellulases to 66%.

Construction of a Bacterial Cellulase Cocktail for Saccharification of Regenerated Cellulose and Pretreated Corn Stover

To apply bacterial cellulases for efficient saccharification of biomass, three Clostridium thermocellum cellulases and a Thermoanaerobacter brockii β-1,4-glucosidase were synthesized in Escherichia coli, and the proportions among them were optimized. When the activities of CelD, CBHA, CBH48Y, and CglT were set at 554, 0.91, 0.91, and 856 mU per assay, respectively, the percent conversion of regenerated cellulose (0.92 g/L) reached 80.9% within 24 h at 60 °C without shaking. Meanwhile, the percent conversion of pretreated corn stover (0.62 g/L) reached 70.1%. Gradually raising the loads of regenerated cellulose from 0.92 to 4.58 g/L resulted in a linear increase in glucose production from 870 to 3208 μg (R2=0.997), as well as a decrease in the percent conversion from 80.9% to 59.6%. These findings suggested that the cellulase cocktail is efficient in saccharification of regenerated cellulose, as well as pretreated corn stover, and has potential applications in the biofuels industry.

A synthetic biology approach for evaluating the functional contribution of designer cellulosome components to deconstruction of cellulosic substrates

BackgroundSelect cellulolytic bacteria produce multi-enzymatic cellulosome complexes that bind to the plant cell wall and catalyze its efficient degradation. The multi-modular interconnecting cellulosomal subunits comprise dockerin-containing enzymes that bind cohesively to cohesin-containing scaffoldins. The organization of the modules into functional polypeptides is achieved by intermodular linkers of different lengths and composition, which provide flexibility to the complex and determine its overall architecture.ResultsUsing a synthetic biology approach, we systematically investigated the spatial organization of the scaffoldin subunit and its effect on cellulose hydrolysis by designing a combinatorial library of recombinant trivalent designer scaffoldins, which contain a carbohydrate-binding module (CBM) and 3 divergent cohesin modules. The positions of the individual modules were shuffled into 24 different arrangements of chimaeric scaffoldins. This basic set was further extended into three sub-sets for each arrangement with intermodular linkers ranging from zero (no linkers), 5 (short linkers) and native linkers of 27–35 amino acids (long linkers). Of the 72 possible scaffoldins, 56 were successfully cloned and 45 of them expressed, representing 14 full sets of chimaeric scaffoldins. The resultant 42-component scaffoldin library was used to assemble designer cellulosomes, comprising three model C. thermocellum cellulases. Activities were examined using Avicel as a pure microcrystalline cellulose substrate and pretreated cellulose-enriched wheat straw as a model substrate derived from a native source. All scaffoldin combinations yielded active trivalent designer cellulosome assemblies on both substrates that exceeded the levels of the free enzyme systems. A preferred modular arrangement for the trivalent designer scaffoldin was not observed for the three enzymes used in this study, indicating that they could be integrated at any position in the designer cellulosome without significant effect on cellulose-degrading activity. Designer cellulosomes assembled with the long-linker scaffoldins achieved higher levels of activity, compared to those assembled with short-and no-linker scaffoldins.ConclusionsThe results demonstrate the robustness of the cellulosome system. Long intermodular scaffoldin linkers are preferable, thus leading to enhanced degradation of cellulosic substrates, presumably due to the increased flexibility and spatial positioning of the attached enzymes in the complex. These findings provide a general basis for improved designer cellulosome systems as a platform for bioethanol production.

Efficient pretreatment for bioethanol production from water hyacinth (eichhornia crassipes) involving naturally isolated and recombinant enzymes and its recovery

Simultaneous saccharification and fermentation (SSF) experiments involving water hyacinth (Eichhornia crassipes), an abundantly available renewable bioresource, using hydrolytic enzymes, and fermentative microbes were investigated. Water hyacinth containing 30.01 (%, w/w) cellulose, 44.49 (%, w/w) hemicellulose, and 20.04 (%, w/w) lignin was subjected to three different pretreatments, namely, wet oxidation, phosphoric acid (H3PO4)‐acetone, and ammonia fiber explosion (AFEX). Hydrolytic enzymes, namely, recombinant Clostridium thermocellum cellulase (GH5) and hemicellulase (GH43), Trichoderma reesei and Bacillus subtilis AS3 cellulases were employed separately for saccharification. Saccharomyces cerevisiae and Candida shehatae were used for fermentation. The AFEX pretreated 1% (w/v) water hyacinth along with recombinant cellulase (GH5)‐hemicellulase (GH43) consortium gave the highest ethanol titer of 1.52 g/L as compared with wet oxidation (1.23 g/L) and phosphoric acid‐acetone pretreatments (1.31 g/L). The best SSF combination with 5% (w/v) substrate at shake flask contributed an ethanol titer and yield of 7.83 g/L, 0.266 (g of ethanol/g of substrate) and its scale up at bioreactor level resulted in significantly higher ethanol titer and yield of 14.39 g/L and 0.489 (g/g), respectively. 93.0 (%, v/v) ethanol from bioreactor was recovered by rotary evaporator with 20.4% purification efficiency. © 2013 American Institute of Chemical Engineers Environ Prog, 33: 1396–1404, 2014

OPTIMIZATION OF AFFINITY DIGESTION FOR THE ISOLATION OF CELLULOSOMES FROM Clostridium thermocellum

The affinity digestion process for cellulase purification consisting of binding to amorphous cellulose, and amorphous cellulose hydrolysis in the presence of dialysis (Morag et al., 1991), was optimized to obtain high activity recoveries and consistent protein recoveries in the isolation of Clostridium thermocellum cellulase. Experiments were conducted using crude supernatant prepared from C. thermocellum grown on either Avicel or cellobiose. While no difference was observed between Avicel-grown or cellobiose-grown cellulase in the adsorption step, differences were observed during the hydrolysis step. The optimal amorphous cellulose loading was found to be 3 mg amorphous cellulose per milligram supernatant protein. At this loading, 90–100% of activity in the crude supernatant was adsorbed. Twenty-four-hour incubation with the amorphous cellulose during the adsorption stage was found to result in maximal and stable adsorption of activity to the substrate. By fitting the adsorption data to the Langmuir model, an adsorption constant of 410 L/g and a binding capacity of 0.249 g cellulase/g cellulose were obtained. The optimal length of time for hydrolysis was found to be 3 hr for cellulase purified from Avicel cultures and 4 hr for cellulase purified from cellobiose cultures. These loadings and incubation times allowed for more than 85% activity recovery.

Structure of CBM4 fromClostridium thermocellumcellulase K

Here, a 2.0 Å resolution X-ray structure of Clostridium thermocellum cellulase K family 4 carbohydrate-binding module (CelK CBM4) is reported. The resulting structure was refined to an R factor of 0.212 and an R(free) of 0.274. Structural analysis shows that this new structure is very similar to the previously solved structure of C. thermocellum CbhA CBM4. Most importantly, these data support the previously proposed notion of an extended binding pocket using a novel tryptophan-containing loop that may be highly conserved in clostridial CBM4 proteins.

Physical association of the catalytic and helper modules of a family-9 glycoside hydrolase is essential for activity

Clostridium thermocellum cellulase 9I (Cel9I) is a non-cellulosomal tri-modular enzyme, consisting of a family-9 glycoside hydrolase (GH9) catalytic module and two family-3 carbohydrate-binding modules (CBM3c and CBM3b). The presence of CBM3c was previously shown to be essential for activity, however the mechanism by which it functions is unclear. We expressed the three recombinant modules independently in Escherichia coli and examined their interactions. Non-denaturing gel electrophoresis, isothermal titration calorimetry, and affinity purification of the GH9-CBM3c complex revealed a specific non-covalent binding interaction between the GH9 module and CBM3c. Their physical association was shown to recover 60–70% of the intact Cel9I endoglucanase activity. Structured summary: MINT- 6946626: Cel9I (uniprotkb: Q02934) and Cel9I (uniprotkb: Q02934) bind (MI: 0407) by comigration in non-denaturing gel electrophoresis (MI: 0404) MINT- 6946649: Cel9I (uniprotkb: Q02934) and Cel9I (uniprotkb: Q02934) bind (MI: 0407) by molecular sieving (MI: 0071) MINT- 6946687: Cel9I (uniprotkb: Q02934) and Cel9I (uniprotkb: Q02934) bind (MI: 0407) by isothermal titration calorimetry (MI: 0065) MINT- 6946706: Cel9I (uniprotkb: Q02934) binds (MI: 0407) to Cel9I (uniprotkb: Q02934) by pull down (MI: 0096)

Bioengineering for pollution prevention through development of biobased energy and materials state of the science report

Industrial BiotechnologyVol. 3, No. 3 Industry ReportBioengineering for pollution prevention through development of biobased energy and materials state of the science reportDianne Ahmann and John R. DorganDianne AhmannSearch for more papers by this author and John R. DorganSearch for more papers by this authorPublished Online:7 Nov 2007https://doi.org/10.1089/ind.2007.3.218AboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail FiguresReferencesRelatedDetailsCited byThe development process of plant-based meat alternatives: raw material formulations and processing strategiesFood Research International, Vol. 3Role of Nanoparticles in Biodegradation and Their Importance in Environmental and Biomedical ApplicationsJournal of Nanomaterials, Vol. 2022Metal oxide nanoparticles for environmental remediationThe development history and recent updates on soy protein-based meat alternativesTrends in Food Science & Technology, Vol. 109BioentrepreneurshipInjection-Molded Parts of Partially Biobased Polyamide 610 and Biobased Halloysite Nanotubes6 July 2020 | Polymers, Vol. 12, No. 7Development of Efficient Composites via Renewable, Recyclable, and Degradable Additives4 December 2019Evaluation of promoter sequences for the secretory production of a Clostridium thermocellum cellulase in Paenibacillus polymyxa26 September 2018 | Applied Microbiology and Biotechnology, Vol. 102, No. 23Rheological properties and microstructure of soy protein isolate /κ-carrageenan gels under high-speed shear treatmentJournal of Food Engineering, Vol. 236Biorenewable blends of polyamide‐4,10 and polyamide‐6,105 April 2016 | Journal of Applied Polymer Science, Vol. 133, No. 45Intellectual Property and Biofuels: The Energy Crisis, Food Security, and Climate ChangeSSRN Electronic JournalOptimal conditions of different flocculation methods for harvesting Scenedesmus sp. cultivated in an open-pond systemBioresource Technology, Vol. 133Next-generation biopolymers: Advanced functionality and improved sustainability16 September 2011 | MRS Bulletin, Vol. 36, No. 9Patenting Trends and Innovation in Industrial BiotechnologySSRN Electronic Journal Volume 3Issue 3Oct 2007 InformationCopyright 2007, Mary Ann Liebert, Inc.To cite this article:Dianne Ahmann and John R. Dorgan.Bioengineering for pollution prevention through development of biobased energy and materials state of the science report.Industrial Biotechnology.Oct 2007.218-259.http://doi.org/10.1089/ind.2007.3.218Published in Volume: 3 Issue 3: November 7, 2007PDF download

Development and characterization of membrane surface display system using molecular chaperon, prsA, of Bacillus subtilis

We report a new membrane surface display system based on molecular chaperon, prsA, of Bacillus subtilis. Clostridium thermocellum cellulase, celA, was fused to C-terminal end of PrsA. Cellulase activity of B. subtilis protoplast, which expressed PrsA–CelA was 15 times higher compared to control strain. More than 85% of total cellulase activity was observed in surface displayed format and less than 15% of total cellulase activity was found in supernatant. Flow cytometric analysis of protoplast of PrsA–CelA fusion expressing bacteria provided another proof of uniform expression of fusion protein onto cytoplasmic membrane of B. subtilis. Without lysozyme treatment, only part of cellulase activity (10%) was observed in whole cell fraction.

Characterization of the subunits in an apparently homogeneous subpopulation of Clostridium thermocellum cellulosomes

Clostridium thermocellum cellulosomes isolated by cellulose affinity chromatography were fractionated by anion exchange chromatography into apparently homogeneous subpopulations that differed with respect to enzyme activity and subunit composition. One such subpopulation contained predominantly six subunits and was closely similar to the “subcellulosome” described by Kobayashi et al. (Kobayashi, T., Romaniec, M. P. M., Fauth, U., and Demain, A. L., Appl. Environ. Microbiol., 1990, 56, 3040–3046). Avicelase specific activity of this homogeneous subpopulation was slightly higher than that of unfractionated cellulosomes, but the two preparations were similarly affected by Ca 2+, dithiothreitol, and cellobiose. Determination of their N-terminal sequences and enzyme activities has enabled three of the six major subunits of the subpopulation of cellulosomes to be positively identified as known components of the C. thermocellum cellulase complex; the other three subunits did not match up with previously characterized cellulosomal proteins.

The resistance of cellulases and xylanases to proteolytic inactivation

The sensitivity of a range of cellulases and xylanases to proteolytic inactivation was investigated. The xylanases, all the Clostridium thermocellum cellulases and cellulase E from Pseudomonas fluorescens subsp. cellulosa exhibited no decrease in catalytic activity during a 3-h incubation with proteinases of the small intestine. Under these conditions, the control Escherichia coli enzymes analysed had half-lives of 4.3-13.5 min. The addition of substrate significantly decreased the sensitivity of proteinase-labile enzymes to inactivation. The significance of these data in relation to the use of cellulases and xylanases for improving animal nutrition is discussed.

Structural features of the Clostridium thermocellum cellulase SS gene.

The Clostridium thermocellum cellulase SS is a subunit of the extracellular cellulase complex (cellulosome). It has previously been shown that SS, hydrolyzes crystalline cellulose synergistically with another subunit, SL. To study this synergism further, the authors cloned the gene coding for SS (celS) and compared its sequence to other known cel genes. The celS, although unique in its DNA sequence, has many structural features similar to those found in other cel genes. These features include a ribosome biding site, signal peptide sequence, the existence of a conserved reiterated amino acid sequence, and a palindromic structure downstream from its open reading frame.

Cloning and DNA sequence of the gene coding for Clostridium thermocellum cellulase Ss (CelS), a major cellulosome component

Clostridium thermocellum ATCC 27405 produces an extracellular cellulase system capable of hydrolyzing crystalline cellulose. The enzyme system involves a multicomponent protein aggregate (the cellulosome) with a total molecular weight in the millions, impeding mechanistic studies. However, two major components of the aggregate, SS (M(r) = 82,000) and SL (M(r) = 250,000), which act synergistically to hydrolyze crystalline cellulose, have been identified (J. H. D. Wu, W. H. Orme-Johnson, and A. L. Demain, Biochemistry 27:1703-1709, 1988). To further study this synergism, we cloned and sequenced the gene (celS) coding for the SS (CelS) protein by using a degenerate, inosine-containing oligonucleotide probe whose sequence was derived from the N-terminal amino acid sequence of the CelS protein. The open reading frame of celS consisted of 2,241 bp encoding 741 amino acid residues. It encoded the N-terminal amino acid sequence and two internal peptide sequences determined for the native CelS protein. A putative ribosome binding site was identified at the 5' end of the gene. A putative signal peptide of 27 amino acid residues was adjacent to the N terminus of the CelS protein. The predicted molecular weight of the secreted protein was 80,670. The celS gene contained a conserved reiterated sequence encoding 24 amino acid residues found in proteins encoded by many other clostridial cel or xyn genes. A palindromic structure was found downstream from the open reading frame. The celS gene is unique among the known cel genes of C. thermocellum. However, it is highly homologous to the partial open reading frame found in C. cellulolyticum and in Caldocellum saccharolyticum, indicating that these genes belong to a new family of cel genes.

Synergism between Clostridium thermocellum cellulases cloned in Escherichia coli.

We have obtained a synergistic effect during degradation of Avicel and filter paper by Clostridium thermocellum cellulases (two endoglucanases and one cellobiohydrolase) cloned in Escherichia coli. The highest degree of synergism was found at early stages of reaction, during the first 20 h: 2.5 and 2.9 on Avicel and filter paper, respectively. During combined action of all three cellulases the main product is cellobiose.

Interaction of the duplicated segment carried by Clostridium thermocellum cellulases with cellulosome components

The function of the non-catalytic, duplicated segment found in C. thermocellum cellulases was investigated. Rabbit antibodies reacting with the duplicated segment of endoglucanase CelD cross-reacted with a variety of cellulosome components ranging between 50 and 100 kDa. 125I-labeled forms of CelD and of xylanase XynZ carrying the duplicated segment bound to a set of cellulosome proteins ranging between 66 and 250 kDa, particularly to the 250 kDa S L (or S1) subunit. 125I-labeled forms of CelD and XynZ devoid of the duplicated segment failed to bind to any cellulosome protein. The duplicated segment appears thus to serve to anchor the various cellulosome subunits to the complex by binding to S L, which may be a scaffolding element of the cellulosome.