Bernard Henrissat

Electron microscopic investigation of the diffusion of Bacillus licheniformis α-amylase into corn starch granules

A method for the direct electron microscopic observation of amylases in interaction with starch granules is presented. The technique involves immuno-gold labeling of the enzymes and cross-sectioning of hydrated starch granules. This approach allows the analysis of the internal degradation of starch with a concomitant visualization of enzymes at the sites of hydrolysis. The visualization of enzymes at the surface, inside the channel and inside the core of the degraded granules shows that the alpha-amylase molecules first proceed from the surface toward the center (centripetal hydrolysis). Then the core is completely degraded from within by erosion of its periphery (centrifugal hydrolysis). In the first case (centripetal hydrolysis), the enzymes act by progressing along the polysaccharide chains. By contrast, the centrifugal hydrolysis leads to even erosion, indicative of a more diffusive motion of the enzymes.

Visualization of the adsorption of a bacterial endo-β-1,4-glucanase and its isolated cellulose-binding domain to crystalline cellulose

Endo-beta-1,4-glucanase A (CenA), a cellulase from the bacterium Cellulomonas fimi, is composed of two domains: a catalytic domain and a cellulose-binding domain. Adsorption of CenA and its isolated cellulose-binding domain (CBD.PTCenA) to Valonia cellulose microcrystals was examined by transmission electron microscopy using an antibody sandwich technique (CenA/CBD.PTCenA-alpha CenA IgG-protein A-gold conjugate). Adsorption of both CenA and CBD.PTCenA occurred along the lengths of the microcrystals, with an apparent preference for certain crystal faces or edges. CenA or CBD.PTCenA, but not the isolated catalytic domain, were shown to prevent the flocculation of microcrystalline bacterial cellulose. The cellulose-binding domain may assist crystalline cellulose hydrolysis in vitro by promoting substrate dispersion.

The crystal structure of methyl β-cellotrioside monohydrate 0.25 ethanolate and its relationship to cellulose II

The crystal structure of methyl beta-cellotrioside (methyl O-beta-D-glucopyranosyl-(1-->4)-O-beta-d_guycopyranosyl-(1-->4)-be ta-D- glucopyranoside) complexed with water and ethanol, C19H34O16. H2O.0.25[C2H6O] was determined by combining Cu K alpha X-ray and synchrotron data collected at room temperature. The crystals have the monoclinic space group P21 with Z = 8 and unit cell parameters a = 7.9978(11), b = 76.38(4), c = 8.9908(6) A and beta = 116.40(1) degree. The structure, which was solved by direct methods and refined to a final R-factor of 0.067, contains four independent molecules of methyl beta-cellotrioside with an extended conformation. They are arranged parallel to the long b axis of the unit cell, and organized in two pairs of antiparallel molecules. Each beta-D-glucopyranosyl residue of the four independent molecules is in the 4C1 pyranose conformation, and each (O-6) primary hydroxyl group has the gt conformation. The crystal structure of methyl beta-cellotrioside has many points in common with that of cellotetraose hemihydrate as well as with the structure of cellulose II. Thus, it is likely that the precise atomic coordinates obtained in this study can be directly transposed to give an improved structure for cellulose II where, in particular, only the gt conformation would be present at the primary hydroxyl groups of both polysaccharide chains.