Versatile derivatives of carbohydrate-binding modules for imaging of complex carbohydrates approaching the molecular level of resolution.

The innate binding specificity of different carbohydrate-binding modules (CBMs) offers a versatile approach for mapping the chemistry and structure of surfaces that contain complex carbohydrates. We have employed the distinct recognition properties of a double His-tagged recombinant CBM tagged with semiconductor quantum dots for direct imaging of crystalline cellulose at the molecular level of resolution, using transmission and scanning transmission electron microscopy. In addition, three different types of CBMs from families 3, 6, and 20 that exhibit different carbohydrate specificities were each fused with either green fluorescent protein (GFP) or red fluorescent protein (RFP) and employed for double-labeling fluorescence microscopy studies of primary cell walls and various mixtures of complex carbohydrate target molecules.

Beta-D-glucosidase reaction kinetics from isothermal titration microcalorimetry.

The cellobiase activities of nine thermal stable mutants of Thermobifida fusca BglC were assayed by isothermal titration microcalorimetry (ITC). The mutations were previously generated using random mutagenesis and identified by high-temperature screening as imparting improved thermal stability to the beta-D-glucosidase enzyme. Analysis of the substrate-saturation curves obtained by ITC for the wild-type enzyme and the nine thermally stabilized mutants revealed that the wild type and all the mutants were subject to binding of a second substrate molecule.

Characterization of thermostable Xyn10A enzyme from mesophilic Clostridium acetobutylicum ATCC 824.

A thermostable xylanase gene, xyn10A (CAP0053), was cloned from Clostridium acetobutylicum ATCC 824. The nucleotide sequence of the C. acetobutylicum xyn10A gene encoded a 318-amino-acid, single-domain, family 10 xylanase, Xyn10A, with a molecular mass of 34 kDa.

Thermostable xylanase10B from Clostridium acetobutylicum ATCC824.

The Clostridium acetobutylicum xylanase gene xyn10B (CAP0116) was cloned from the type strain ATCC 824, whose genome was recently sequenced. The nucleotide sequence of C. acetobutylicum xyn10B encodes a 318-amino acid protein.

Essential role of the family-22 carbohydrate-binding modules for beta-1,3-1,4-glucanase activity of Clostridium stercorarium Xyn10B.

Clostridium stercorarium Xyn10B is a modular enzyme comprising two family-22 carbohydrate-binding modules (CBMs), a family-10 catalytic module of glycoside hydrolases, a family-9 CBM, and two S-layer homologous modules consecutively from the N-terminus. To investigate the role of the family-22 CBMs, truncated proteins were constructed: a recombinant catalytic module polypeptide (rCD), a CBM polypeptide composed of two family-22 CBMs (rCBM) and a polypeptide composed of the family-22 CBMs and the catalytic module (rCBM-CD). We found that rCBM-CD was highly active toward beta-1,3-1,4-glucan; however, rCD was negligibly active toward the same substrate.