Search

The digestion of bacterial cellulose ribbons by ternary mixtures of enzymes consisting of recombinant cellulases (two cellobiohydrolases, Cel6A and Cel7A, and the endoglucanase Cel45A) from Humicola insolens was investigated over a wide range of mixture composition. The extent of digestion was followed by soluble sugar release (saccharification) analysis together with transmission electron microscopy (TEM) observations. It was found that the addition of minute quantities of Cel45A induced a spectacular increase in saccharification of the substrate with either Cel7A or the mixture of Cel6A and Cel7A. Conversely, only a moderate saccharification resulted from the mixing of Cel45A and Cel6A. This difference is believed to originate from (1) the occasional endo character of Cel6A and (2) the competition of Cel6A and Cel45A for the substrate sites that are sensitive to endo activity. Interestingly, the mixture of enzymes giving rise to the highest saccharification rate did not always correspond to mixtures of enzymes generating the highest synergy. TEM images revealed that the bacterial cellulose ribbons became at the same time cut and narrowed down under the action of an optimized mixture of the three enzymes. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 72: 339–345, 2001.

The action of cellulosomes from Clostridium thermocellum on model cellulose microfibrils from Acetobacter xylinum and cellulose microcrystals from Valoniaventricosa was investigated. The biodegradation of these substrates was followed by transmission electron microscopy, Fourier-transform IR spectroscopy and X-ray diffraction analysis, as a function of the extent of degradation. The cellulosomes were very effective in catalysing the complete digestion of bacterial cellulose, but the total degradation of Valonia microcrystals was achieved more slowly. Ultrastructural observations during the digestion process suggested that the rapid degradation of bacterial cellulose was the result of a very efficient synergistic action of the various enzymic components that are attached to the scaffolding protein of the cellulosomes. The degraded Valonia sample assumed various shapes, ranging from thinned-down microcrystals to crystals where one end was pointed and the other intact. This complexity may be correlated with the multi-enzyme content of the cellulosomes and possibly to a diversity of the cellulosome composition within a given batch. Another aspect of the digestion of model celluloses by cellulosomes is the relative invariability of their crystallinity, together with their Iα/Iβ composition throughout the degradation process. Comparison of the action of cellulosomes with that of fungal enzymes indicated that the degradation of cellulose crystals by cellulosomes occurred with only limited levels of processivity, in contrast with the observations reported for fungal enzymes. The findings were consistent with a mechanism whereby initial attack by a cellulosome of an individual cellulose crystal results in its ‘commitment’ towards complete degradation.

Heterobifunktionelle Liganden, die gleichzeitig an die Katalysedomäne und an die Stärkebindungsdomäne von Glucoamylase binden, induzieren eine Konformationsänderung des Proteins, wie mit dynamischer Lichtstreuung gezeigt wurde. Die Liganden bestehen aus Acarbose und β-Cyclodextrin, die über Oligoethylenglycol-Linker unterschiedlicher Länge miteinander verbunden sind (siehe schematische Darstellung).

The molecular packing in β-chitin unit cells was experimentally determined by a combination of unidirectional degradation by Bacillus circulans chitinase A1 and microdiffraction electron crystallography using highly crystalline β-chitin microfibrils from the protective tubes secreted by Lamellibrachia satsuma. The mode of chain packing was found to be identical with that of the previously published crystal model for β-chitin, despite a controversial definition of the unit cell parameters. Here, a “parallel-down” packing was determined, where the reducing ends of chains point in an opposite direction to the crystallographic c-axis. Microdiffraction analyses of nascent β-chitin microfibrils generated from diatom Thalassiosira sp. showed that the c-axis of the crystal was directed toward the diatoms, and therefore the reducing end of a growing chain pointed away from the locus of biosynthesis. This mechanism agreed well with what we found recently in the cellulose biosynthesis system, and provides strong evidence that the polymerization by the processive glycosyl transferase takes place at the non-reducing end of the growing polysaccharide chains.

Heterobifunctional ligands that bind at the same time to the catalytic domain and to the starch-binding domain of glucoamylase induce a conformational change of the protein, as shown by dynamic light scattering. The ligands consist of acarbose and β-cyclodextrin linked together by oligoethylene glycols of variable length (see the schematic diagram).

On the basis of the ‘parallel‐up' structure of the cellulose crystal, a crystallographic approach to study the mode of action of cellobiohydrolase Cel7A on Valoniacellulose microcrystal has been carried out. After incubation with Cel7A, most of the initially smooth and well defined Valoniamicrocrystals displayed fibrillation. However, as the hydrolysis reaction was rather heterogeneous, some microcrystals remained superficially intact. Close investigation on such crystals revealed polar morphology: one end was narrowed extremely or pointed. Electron microdiffraction analysis of these crystals evidenced that the narrowing of the microcrystals occurs at their reducing end side. This was also confirmed by the visualization of selective reducing end labeling at the pointed ends of microcrystals. These lines of investigation are the first demonstration that the processivity of Cel7A action against insoluble highly crystalline celluloses is unambiguously toward non‐reducing ends from reducing ends.

Endoglucanase V (EG V) of Humicola insolensis composed of a catalytic domain and of a cellulose-binding domain linked by a 33 amino acid long peptide rich in Ser, Thr and Pro residues. This work describes the dynamic behavior of the two-domain structure of EG V as revealed by quasi-elastic light scattering experiments. For both the full-length and the isolated catalytic domain, the autocorrelation function is essentially described by a single relaxation mode. The equivalent hydrodynamic radius of the catalytic domain was found to correspond precisely to the dimensions measured from the previously determined three-dimensional structure. The results obtained with the full-length protein allow a description of the two domain structure of EG V similar to that resulting from earlier studies using small angle X-ray scattering on cellulases from Trichoderma reesei. The hydrodynamic dimensions of the entire enzyme can be approximated as an ellipsoid with dimensions of 42 × 133.6 Å.

Endoglucanase V (EG V) of Humicola insolensis composed of a catalytic domain and of a cellulose‐binding domain linked by a 33 amino acid long peptide rich in Ser, Thr and Pro residues. This work describes the dynamic behavior of the two‐domain structure of EG V as revealed by quasi‐elastic light scattering experiments. For both the full‐length and the isolated catalytic domain, the autocorrelation function is essentially described by a single relaxation mode. The equivalent hydrodynamic radius of the catalytic domain was found to correspond precisely to the dimensions measured from the previously determined three‐dimensional structure. The results obtained with the full‐length protein allow a description of the two domain structure of EG V similar to that resulting from earlier studies using small angle X‐ray scattering on cellulases from Trichoderma reesei. The hydrodynamic dimensions of the entire enzyme can be approximated as an ellipsoid with dimensions of .

Because cellulases are finding more applications in the textile and detergent industries, their effect on cotton fibres must be evaluated. For this purpose, the action of a recombinant cellulase, endoglucanase V from the fungus Humicola insolens, has been followed by scanning electron microscopy (SEM) in classical longitudinal views as well as in cross-sections of cotton fibres. The experiments were conducted at large enzyme dilution typical of conditions where cellulases are used for biopolishing, i.e. for the removal of defects created by mechanical abrasion. Endoglucanase V appears to restrict its action to the hydrolysis of the loose fibrils created at the surface of the fibres and no indication of extensive enzyme penetration and damage to the interior of the fibres could be detected by SEM. The adsorption sites for endoglucanase V on cotton fibres were examined by transmission electron microscopy (TEM) on ultrathin cross-sections after immuno-gold labeling of the enzyme. This approach showed that the enzymes did not penetrate the fibres but remained at their surface. The use of an immuno-gold labeled cellulase provides a new way to probe the surface features of cotton fibres

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.