Clostridium thermocellum

Clostridium acetobutylicum p262 Clostridium acetobutylicum ATCC824 Clostridium pasteurianum Clostridium perfringens Clostridium thermocellum... 

Within the cellulosome complex, type I dockerin domain is responsible for incorporating its associated glycosyl hydrolase in the bacterial cellulosome via interaction with a reception domain, the cohesin domain. The three-dimensional solution structure of the 69-residue dockerin domain from the thermophilic Clostridium thermocellum (Topt = 55-65 °C) was solved by NMR and was found to consist of two Ca " -binding loop-helix motifs connected by a linker. Each Ca " -binding subdomain is stabilized by a cluster of buried hydrophobic sidechains. Recently, the NMR sequence-specific resonance assignment of type II cohesin module from C. thermocellum has been published. ... 

Our early studies dealt with characterization of cellulase from Clostridium thermocellum (4, 5), the first described thermoanaerobe. More recently, we have characterized the saccharidases in three new non-cellulolytic thermoanaerobic species (6-12). Table II compares the general properties of thermophilic saccharidases identified in C. thermosulfurogenes strain 4B (6), C. thermohydrosulfuricum strain 39E (7), and Thermoanaerohacter strain B6A (13). It is worth noting here that... 

Clostridium thermocellum Cellulase D (27) Pseudomonas fluorescens CMCase (22)... 

Figure 1. The organization of catalytic and non-catalytic domains in cellulases from C. fimi and other bacteria. CfCenA, B and C, and CfCex are the endo- and exo-p- 1, 4-glucanases of C. fimi, ClfX is a translated open reading frame from Cellulomonas flavigena (29), CtEGD and PfEndA are endo-p-1, 4-glucanases from Clostridium thermocellum and Pseudomonas fluorescens, respectively (30,31), The primary structures are drawn approximately to scale and are numbered from the amino terminus of the mature protein ClfX is numbered from the start of the open reading frame. Unshaded areas represent catalytic domains, cross-hatched areas indicate cellulose-binding domains, repeated blocks of amino acids are stippled, and black areas represent linker regions.

Catalytic Domains. The catalytic domain of CenB (amino acids 1 - 608) shows 35% sequence identity with an endoglucanase from Persea americana (avocado) it has therefore been placed in family E, subfamily 2 (2,3). The CenC catalytic domain (amino acids 299-809) shows 28% sequence identity with Clostridium thermocellum endoglucanase D (EGD) and 43% identity with P. fluorescens endoglucanase A (End A), both members of family E, subfamily 1 (2,3). 

Clostridium thermocellum endoglucanase CelE PLVS(PT)3LMPTPSPTVT 37... 

Clostridium thermocellum xylanase Z active on 4-methylumbelliferyl p-D-cellobioside 52... 

Figure 1. Amino acid sequences of microbial glycohydrolases. A Aureobasid-iwn sp. endo-xylanase Sc Schizophyllwn commune endo-xylanase C Chainia sp. endo-xylanase Bp Bacillus pumilus endo-xylanase Bs Bacillus subtilis Bacillus circulans endo-xylanase Pf Pseudomonas fluorescens endo-xylanase B alkalophilic Bacillus sp. endo-xylanase Ct Clostridium thermocellum endo-xylanase Cf Cellulomonas fimi cellobiohydrolase Ca Cryptococcus albidus endo-xylanase. Residue numbers are those of the adjacent residue, counting from the N-terminus of the mature protein.

Figure 2. Specificities of Endoglucanases (EGA, EGB, EGC, EGD) from Clostridium thermocellum cloned in E. coli (10). The substrates (MeUmb-Glc , n = 2-5, MeUmbLac) are depicted (symbols A, (3-1,4 galactopyra-nosyl , / -1,4 glucopyranosyl , 4-methylumbelliferyl) and the arrows indicate scission points as determined by HPLC (1).

Figure 6. Affinity chromatography of EGD from Clostridium thermocellum. Nucleic acid preparation, heat treatment and ammonium sulfate precipitation (0-70%, 70-100%) were carried out as described (10). The final precipitate ( 50 mg protein), dissolved in 50 mM sodium acetate, pH 5.0, was applied (after centrifugation) on the affinity column (2 x 25 cm) (4 -aminobenzyl l-thio-/ -cellobioside coupled to Sepharose 4B) (11). Protein was monitored at 280 nm and the activity of the fractions (2 ml) determined using 2 -chloro-4 -nitrophenyl / -cellobioside (pH 6.5, 25°C) as described in the text. Elution with 10 mM G2 was started as indicated.

The T. reesei enzymes could also be cleaved into separate domains by proteolysis, and this was discussed elsewhere (Claeyssens, M., and Tomme, P., this volume). Suffice it to emphasize here that the genes encoding cellulases in C. fimi and T. reesei appear to have arisen by domain shuffling and that the enzymes they encode appear to interact with cellulose in a comparable manner, i.e., a catalytic domain is held on the substrate by a binding domain. Cellulases from the bacterium Clostridium thermocellum also contained sequences analogous to Pro-Thr boxes as well as highly conserved carboxyl terminal sequences (2,17,18). It remains to be seen if they have functional organizations similar to those of the C. fimi and T. reesei enzymes. 

Similar approaches to cloning of bacterial xylanases have been used for genes from Clostridium acetobutylicum (30), Bacillus polymyxa (31), Bacteroides succinogenes (32), Clostridium thermocellum (33) and Pseudomonas fluorescens subsp. cellulosa (34). In each case the xylanases were predominantly located intracellularly and the levels of xylanases produced from cloned systems were, in general, very low in comparison to yeast and fungal systems. A comparison of the production yields and extent of extracellular production for various cloned xylanase genes is found in Table... 

Ethanol Saccharomyces cerevisiae, Zymomonas mobilis, Clostridium thermocellum and other Clostridium spp. Alcoholic beverages solvent in chemical industry fuel extender... 

Many fungi are capable of producing extracellular enzymes that can degrade cellulose. They are Trichoderma (T) reesei, T. viride, T. koningii, T. lignorum, Penicillium funiculosum, Fusarium solani, Sclerotium rolfsii, and so on. Bacterial species such as Cellulomonas along with Clostridium thermocellum can also produce cellulases (Marsden and Gray, 1986). 

Clostridium thermocellum Anaerobe Metabolic labeling Metabolic pathway analysis (175)... 

Lamed, R. I, Lobos, J. H., Su,T. M. 1988. Effects of stirring and hydrogen on fermentation products of Clostridium thermocellum. Appl. Environ. Microbiol, 54, 1216-1221. 

We next tried to provide a-GlP, not from sucrose, but from cellobiose by using CBP. When CBP from Clostridium thermocellum (Kim et al., 2002) and GP were incubated with cellobiose in the presence of Pi and maltooligosac-chride primer, linear a-1,4 glucan was synthesized wth a yield (38.6 %) that was much lower than in the SP-GP method (Ohdan et al., 2007). To improve the yield of amylose, mutarotase and glucose oxidase were added to the initial reaction mixture with the expectation that they would remove the glucose derived from the CBP reaction and consequently shift the equilibrium state to phosphorolysis. The yield of amylose was increased to 64.8% (Ohdan et al., 2007). 

Kim, Y. K., Kitaoka, M., Krishnareddy, M., Mori, Y., and Hayashi, K. 2002. Kinetic studies of a recombinant cellobiose phosphorylase (CBP) of the Clostridium thermocellum YM4 strain expressed in Escherichia coli. J. Biochem., 132, 197-203. 

Zhang, Y.-H. P. and Lynd, L. R., Cellulose utilization by Clostridium thermocellum Bioenergetics and hydrolysis product assimilation. PNAS 2005, 102 (20), 7321-7325. 

Johnson, E. A., Sakajoh, M., Halliwell, G., Madia, A., and Demain, A. L., Saccharification of complex cellu-losic substrates by the cellulase system from Clostridium thermocellum. Appl. Environ. Microbiol. 1982, 43, 1125-1132. 

An efficient system for the production of recombinant antibodies is cellulose-assisted refolding technology, as described by Berdichevsky et al. [7]. The expressed scFvs were fused to a cellulose-binding domain (CBD) from the bacterium Clostridium thermocellum in the format scFv-CBD. The resulting fusion proteins were obtained in high yield from bacterially produced inclusion bodies that become solubilized and then refold while immobilized on cellulose. The refolded and purified scFv-CBD fusion proteins can be used to form cellulose-based affinity matrices or, as described herein, can be immobilized on a cellulose matrix that makes up part of the immunoelectrochemical sensor device. 

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