Clostridium thermocellum, cellulase

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

Kurokawa J, Hemjinda E, Aral T, Kimura T, Sakka K, Ohmiya K (2002) Clostridium thermocellum cellulase CelT, a family 9 endoglucanase without an Ig-like domain or family 3c carbohydrate-binding module. Appl Microbiol Biotechnol 59 455-461... 

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... 

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.

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. 

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). 

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. 

The cellulolytic system from culture filtrates of Clostridium thermocellum, when partially purified by preparative electrophoresis, showed a single band on analytical polyacrylamide gel electrophoresis.However, sodium dodecyl sulphate polyacrylamide gel electrophoresis indicated that at least five proteins were present. Neutral carbohydrate was present in the purified fraction and it was suggested that the cellulase was present as a complex. 

Zhang, Y. and Lynd, L.R. (2003) Quantification of cell and cellulase mass concentrations during anaerobic cellulose fermentation development of an enzyme-linked immunosorbent assay-based method with application to Clostridium thermocellum batch cultures. Anal. Chem., 75 (2), 219—227. 

In direct microbial conversion of lignocellulosic biomass into ethanol that could simplify the ethanol production process from these materials and reduce ethanol production costs, Clostridium thermocellum, a thermoanaerobe was used for enzyme production, hydrolysis and glucose fermentation (755). Cofermentation with C thermosaccharolyticum simultaneously converted the hemicellulosic sugars to ethanol. However, the formations of by-products such as acetic acid and low ethanol tolerance are some drawbacks of the process. Neurospora crassa produces extracellular cellulase and xylanase and has the ability to ferment cellulose to ethanol 139). 

Another thermophilic bacterium, Clostridium thermocellum, possesses a distinct cellulase activity that requires the presence of Ca and a reducing agent, and is more active against crystalline cellulose than amorphous cellulose (12). This particular cellulase exists as a cellulosome, an enormous complex that contains at least 15 different proteins (13), all of which have been cloned and sequenced... 

The synthesis of extracellular cellulase activity by three strains of Clostridium thermocellum growing on cellulose has been shown to be directly related to the degradation of cellulose and to the growth of the bacteria. Cellulase was not detected when the cells grow on cellobiose. The activities of the exo-D-glucanase (pH optimum 5.2, temperature optimum 65 °C) and e do-D-glucanase (pH optimum 5.4, temperature optimum 64 °C) were compared. o-Glucose and cellobiose were released when the crude cellulase acted on cellulose, cellotetraose, and cellotriose in vitro. 

Cornet P, Millet J, Beguin P, Aubert J P 1983 Characterization of two cell (cellulase degradation) genes of Clostridium thermocellum coding for endoglucanases. Bio/Tfechnology 1 589-594... 

Wood BE, Ingram LO (1992) Ethanol production from cellobiose, amorphous cellulose, and crystalline cellulose by recombinant Klebsiella oxytoca containing chromosomally integrated Zymomonas mobilis genes for ethanol production and plasmids expressing thermostable cellulase genes from Clostridium thermocellum. Appl Environ Microbiol 58 2103-2110... 

Cellulase complexes similar to the cellulosomes of C. thermocellum were also identified in other cellulolytic bacteria such as the mesophilic species C. cellulovorans, C. cellulolyticum, C. papyrosolvens, Bacteroides cellulosolvens, Acetivibrio cellulolyticus,Ruminococcus albus, and Ruminococcus flavefaciens and the thermophilic species Clostridium clariflavum and Clostridium josui. They... 

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