[The family 28 carbohydrate-binding module of the thermostable endo-1,4-beta-glucanase CelD Caldicellulosiruptor bescii maximizes the enzyme's activity and binds irreversibly to amorphous cellulose].
The nucleotide sequence of a chromosome fragment of the thermophilic anaerobic bacterium Caldicellulosiruptor bescii (syn. Anaerocellum thermophilum) has been determined. The fragment contains four open reading frames with the second one of 749 aa encoding a multimodular endo-1,4-beta-glucanase CelD (85019 Da). N-terminal region of the protein includes the signal peptide and the catalytic module of glycoside hydrolase family 5 (GH5), followed by the substrate-binding module of family 28 (CBM28). The C-terminal region bears three SLH modules. The recombinant endoglucanase and its two separate modules, the catalytic one and CBM28, were produced in E. coli cells and purified to homogeneity. Analysis of the catalytic properties showed CelD to be endo-1,4-beta-glucanase whose maximum activity was exhibited on beta-glucan of barley at pH 6.2 and 70 degrees C. The enzyme was stable at 50 degrees C for 30 days. Upon removal of the C-terminal CBM28, the activity of GH5 decreased on cellulose substrates, and its thermostability was dropped. Binding of CBM28 to amorphous cellulose was almost irreversible as it could not be removed from this substrate in a range of pH 4-11, temperatures--of 0-75 degrees C, and NaCl concentration--of 0-5 M. Only 100% formamide or 1% SDS were able to remove the protein.
G A
GA
Velikodvorskaia
L A
LA
Chekanovskaia
N A
NA
Lunina
O V
OV
Sergienko
V G
VG
Lunin
I A
IA
Dvortsov
V V
VV
Zverlov
D001419:Bacteria / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002480:Cellulase / Q000235:genetics / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D016680:Genome, Bacterial; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013696:Temperature
D011134:Polysaccharides; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D002482:Cellulose; C087879:endoglucanase CelD; D002480:Cellulase