The cellulose‐binding domain (CBD_Cex) of an exoglucanase from Cellulomonas fimi: Production in Escherichia coli and characterization of the polypeptide

Abstract

The gene fragment encoding the cellulose-binding domain (CBD) of an exoglucanase (Cex) from Cellulomonas fimi was subcloned and expressed in Escherichia coli. Transcription from the lac promoter coupled with translation from a consensus prokaryotic ribosome binding site led to the production of large quantities of CBD(Cex) (up to 25% total soluble cell protein). The polypeptide leaked into the culture supernatant (up to 50 mg . L(-1)), facilitating one-step purification by affinity chromatography on cellulose. The 11-kDa polypeptide reacted with Cex antiserum. Absence of free thiols indicated that the two Cys residues of CBD(Cex) form a disulfide bridge. It had the same N-terminal amino acid sequence as CBD(Cex) prepared from Cex by proteolysis, plus two additional N-terminal amino acid residues (Ala and Ser) encoded by the Nhel site introduced during plasmid construction. CBD(Cex) bound to a variety of beta-1, 4-glycans with different affinities and saturation levels. Adsorption to bacterial microcrystalline cellulose was dependent on the temperature, but not on the pH.

Direct Demonstration of the Flexibility of the Glycosylated Proline-Threonine Linker in the Cellulomonas fimi Xylanase Cex through NMR Spectroscopic Analysis

“…Thus, neither the linker nor the cellulose-binding module alters the catalytic properties of the catalytic domain of Cex. Likewise, the relative binding affinities of nonglycosylated Cex and the isolated CexCBD for microcrystalline cellulose (Avicel) are very similar, indicating that the catalytic domain and linker neither contribute to, nor interfere with, cellulose binding (55). Complementing these findings, thermal denaturation studies also revealed that the stabilities of the catalytic domain and cellulose-binding modules of Cex do not change significantly upon their separation (56).…”

The Use of Carbohydrate Binding Modules (CBMs) to Monitor Changes in Fragmentation and Cellulose Fiber Surface Morphology during Cellulase- and Swollenin-induced Deconstruction of Lignocellulosic Substrates

“…The procedure used previously with CEL61A (34) was followed. CBM2a from Cellulomonas fimi was purified from Escherichia coli using the following procedure (35). Cells were grown at 37°C in Terrific Broth to A 280 ϳ2.0 and then induced with 0.2 mM isopropyl-␤-D-1-thiogalactopyranoside (Sigma-Aldrich) and incubated for an additional 24 -32 h at 30°C.…”

“…The outliers in the adsorption profile of the CBMs are the fungal family 1 CBMs (21, 31, 49) as these small (ϳ4 kDa, as compared with 27 kDa for CBM44 and 11 kDa for CBM2a (30,35)) Type A fungal enzyme-derived CBMs are known to preferentially bind to dislocations. These CBMs, which have the highest binding order parameters of all CBMs studied to date (indicating a high preference for crystalline cellulose (48)), appear to localize at dislocations.…”

Probing the role of tryptophan residues in a cellulose‐binding domain by chemical modification

“…NMR spectra were obtained with a Varian Unity 500-MHz spectrometer equipped with a pulsed field gradient triple resonance probe, with an actively shielded z gradient coil, as described previously (Xu et al, 1995). CBDcex binding to BMCC was assayed using individual samples as described previously (Ong et al, 1993), except that mixtures were incubated for I h at 4 "C instead of overnight. Residual binding was calculated from the difference between pairs of samples incubated for the same times with and without added NBS.…”