celB, a gene coding for a bifunctional cellulase from the extreme thermophile "Caldocellum saccharolyticum"

Abstract

Caldocellum saccharolyticum" is an obligatory anaerobic thermophilic bacterium. A gene from this organism, designated celB, has been cloned in Eschenichia coli as part of a bacteriophage A gene library. This gene produces a thermostable cellulase that shows both endoglucanase and exoglucanase activities on test substrates and is able to degrade crystalline cellulose to glucose. The sequence of celB has homology with both exo-and endoglucanases described by others. It appears to have a central domain without enzymatic activity which is joined to the enzymatic domains by runs of amino acids rich in proline and threonine (PT boxes). Deletion analysis shows that the exoglucanase activity is located in the amino-terminal domain of the enzyme and that endoglucanase activity is located in the carboxy-terminal domain. There are internal transcriptional and translational start sites within the gene. The intact gene has been cloned into a temperature-inducible expression vector, pJLA602, and overexpressed in E. coli. Polyacrylamide gel electrophoresis showed that celB produced a protein with a molecular weight of 118,000 to 120,000. A number of smaller proteins with activity against carboxymethyl cellulose and 4-methyl umbelliferyl-o-D-cellobioside were also produced. These are believed to be the result of alternative translational start sites and/or proteolytic degradation products of the translated gene product. The cellulase system comprises three general classes of enzymes: exoglucanases (P-1,4-D-glucan cellobiohydrolase), endoglucanases (P-1,4-D-glucan glucanhydrolase), and

Thermophilic lignocellulose deconstruction

“…Large, multimodular carbohydrate‐active enzymes were discovered to be the distinguishing feature of the genus Caldicellulosiruptor , with CelB (Table ) being the first described modular multifunctional enzyme (Saul et al ., ), followed by the sequencing of other modular enzymes, such as manA (Gibbs et al ., ) and celA (Te'O et al ., ). Incidentally, although discovered almost 20 years ago, CelA remains one of the largest discrete cellulase proteins known (1751 amino acids, predicted molecular weight of 194.8 kDa).…”

“…The third extracellular module, GH5c, is also found as a single module in multifunctional, modular enzymes paired with xylanases (GH10; VanFossen et al ., ) or β‐mannanases (GH5b). In support of the intragenic recombination hypothesis, a linker segment from a modular GH5c enzyme (Csac_1078) was found to be longer (van de Werken et al ., ; VanFossen et al ., ) than previously reported (Saul et al ., ), and orthologs of this enzyme will vary in the number of CBM3s observed (one, Csac_1078; two, COB47_1671; and three, sp. Tok7B.1).…”

Induction of production and secretion β(1→4) glucanase withSaccharomyces cerevesiaeby replacing theMET10gene withegl1gene fromTrichoderma reesei

“…SDS-PAGE was performed in gels containing 10% (w ⁄ v) acrylamide and 0AE1% (w ⁄ v) SDS using a Tris ⁄ glycine buffer (pH8AE5) system. The method of Saul (Saul et al 1990) was modified as followings: cell-free extracts protein (70 lg) was separated on a 10% polyacrylamide gel with 0AE15% CMC-Na included in the separating layer. Sodium dodecylsulfate was removed by washing solution in four changes.…”

Genomic and physiological analyses reveal that extremely thermophilicCaldicellulosiruptor changbaiensisdeploys unique cellulose attachment mechanisms

“…The original isolates from the genus Caldicellulosiruptor were identified on the basis of their ability to grow on cellulose at elevated temperatures [56,54], especially temperatures beyond the optimal growth temperature of Ruminiclostridium thermocellum [48]. Interest in thermostable enzymes produced by this genus continues, as the initial discovery of their multifunctional, modular enzymes [51,26,57,67] represented an alternate paradigm to cellulosomes [2,52]. Further discoveries on the capabilities of these thermostable enzymes include the unique mode of action used by the central cellulase, CelA, [8], synergistic activity in ionic liquid optimized enzyme mixtures [45,46] and the creation of designer cellulosomes from Caldicellulosiruptor catalytic domains [29].…”