Solubilization of cellulosomal cellulases by fusion with cellulose-binding domain of noncellulosomal cellulase engd from Clostridium cellulovorans

ABSTRACT The planar and anchoring residues of the family IIIa cellulose binding domain (CBD) from the cellulosomal scaffolding protein of Clostridium cellulovorans were investigated by site-directed mutagenesis and cellulose binding studies. By fusion with maltose binding protein, the family IIIa recombinant wild-type and mutant CBDs from C. cellulovorans were expressed as soluble forms. Cellulose binding tests of the mutant CBDs indicated that the planar strip residues played a major role in cellulose binding and that the anchoring residues played only a minor role.

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Characterization of EngF from Clostridium cellulovorans and Identification of a Novel Cellulose Binding Domain

Applied and Environmental Microbiology ◽

10.1128/aem.64.3.1086-1090.1998 ◽

1998 ◽

Vol 64 (3) ◽

pp. 1086-1090 ◽

Cited By ~ 14

Author(s):

Akihiko Ichi-ishi ◽

Salah Sheweita ◽

Roy H. Doi

Keyword(s):

Amino Acids ◽

Cellulose Degradation ◽

Binding Activity ◽

Binding Domain ◽

Cellulose Binding Domain ◽

Binding Studies ◽

Endoglucanase Activity ◽

Clostridium Cellulovorans ◽

C Terminus ◽

Cellulose Binding

ABSTRACT The physical and enzymatic properties of noncellulosomal endoglucanase F (EngF) from Clostridium cellulovorans were studied. Binding studies revealed that the Kd and the maximum amount of protein bound for acid-swollen cellulose were 1.8 μM and 7.1 μmol/g of cellulose, respectively. The presence of cellobiose but not glucose or maltose could dissociate EngF from cellulose. N- and C-terminally truncated enzymes showed that binding activity was located at some site between amino acid residues 356 and 557 and that enzyme activity was still present when 20 amino acids but not 45 amino acids were removed from the N terminus and when 32 amino acids were removed from the C terminus; when 57 amino acids were removed from the C terminus, all activity was lost. EngF showed low endoglucanase activity and could hydrolyze cellotetraose and cellopentaose but not cellotriose. Activity studies suggested that EngF plays a role as an endoglucanase during cellulose degradation. Comparative sequence analyses indicated strongly that the cellulose binding domain (CBD) is different from previously reported CBDs.

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Cellulose as an inert matrix for presenting cytokines to target cells: production and properties of a stem cell factor–cellulose-binding domain fusion protein

Biochemical Journal ◽

10.1042/bj3390429 ◽

1999 ◽

Vol 339 (2) ◽

pp. 429-434

Author(s):

J. Greg DOHENY ◽

Eric J. JERVIS ◽

M. Marta GUARNA ◽

R. Keith HUMPHRIES ◽

R. Antony J. WARREN ◽

...

Keyword(s):

Stem Cell ◽

Fusion Protein ◽

Stem Cell Factor ◽

Direct Analysis ◽

Binding Domain ◽

Target Cells ◽

Cellulose Binding Domain ◽

Binding Characteristics ◽

Cellulose Binding ◽

Cellulose Surface

A chimaera of stem cell factor (SCF) and a cellulose-binding domain from the xylanase Cex (CBDCex) effectively immobilizes SCF on a cellulose surface. The fusion protein retains both the cytokine properties of SCF and the cellulose-binding characteristics of CBDCex. When adsorbed on cellulose, SCF–CBDCex is up to 7-fold more potent than soluble SCF–CBDCex and than native SCF at stimulating the proliferation of factor-dependent cell lines. When cells are incubated with cellulose-bound SCF–CBDCex, activated receptors and SCF–CBDCex co-localize on the cellulose matrix. The strong binding of SCF–CBDCex to the cellulose surface permits the effective and localized stimulation of target cells; this is potentially significant for long-term perfusion culturing of factor-dependent cells. It also permits the direct analysis of the effects of surface-bound cytokines on target cells.

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