Water-insoluble Cellulose Research Articles

Purification and characterization of an endo-1,4-beta-glucanase from Neisseria sicca SB that hydrolyzes beta-1,4 linkages in cellulose acetate.

An enzyme catalyzing hydrolysis of beta-1,4 bonds in cellulose acetate was purified 18.3-fold to electrophoretic homogeneity from a culture supernatant of Neisseria sicca SB, which can assimilate cellulose acetate as the sole carbon and energy source. The molecular mass of the enzyme was 41 kDa and the isoelectric point was 4.8. The pH and temperature optima of the enzyme were 6.0-7.0 and 60 degrees C. The enzyme catalyzed hydrolysis of water-soluble cellulose acetate (degree of substitution, 0.88) and carboxymethyl cellulose. The Km and Vmax for water-soluble cellulose acetate and carboxymethyl cellulose were 0.242% and 2.24 micromol/min/mg, and 2.28% and 12.8 micromol/min/mg, respectively. It is estimated that the enzyme is a kind of endo-1,4-beta-glucanase (EC 3.2.1.4) from the substrate specificity and hydrolysis products of cellooligosaccharides. The enzyme and cellulose acetate esterase from Neisseria sicca SB degraded water-insoluble cellulose acetate by synergistic action.

Optimization of monosaccharide recovery by post-hydrolysis of the water-soluble hemicellulose component after steam explosion of softwood chips

SO 2-catalyzed steam explosion effectively fractionates softwood carbohydrates, releasing 80–90% of the hemicellulose into solution and enhancing the enzymatic hydrolysis of the water-insoluble cellulose fraction. When steam pretreatment conditions were modified to ensure maximum hemicellulose recovery, a significant part of hemicellulose-derived carbohydrates were solubilized in an oligomeric form that was not readily fermented. When Douglas fir wood chips were used as the substrate, sulfuric acid-catalyzed post-hydrolysis at 120°C for 1 h allowed most of the original hemicellulose to be recovered in the monomeric form. Although higher catalyst concentrations (1–3%) resulted in complete depolymerization of the carbohydrates, noticeable losses due to sugar degradation were observed. Lower concentrations of the added catalyst increased the monomer yields, and resulted in less secondary degradation. Partial oxidation of the added SO 2 to sulfuric acid during steam explosion resulted in the depolymerization of the hemicelluloses during post-hydrolysis, even without further acid addition. Hexoses present in the post-hydrolyzates could be readily fermented to ethanol.

Dietary Effect of Guar Gum and its Partially Hydrolyzed Product on the Lipid Metabolism and Immune Function of Sprague-Dawley Rats

The dietary effect of the water-soluble dietary fibers (WSDF), guar gum, partially hydrolyzed guar gum (PHGG), glucomannan, highly methoxylated (HM) pectin, on the serum lipid level and immunoglobulin (Ig) production of Sprague-Dawley rats was compared with that of water-insoluble cellulose. Although serum total cholesterol and triglyceride levels were significantly lower in the rats fed with WSDF than in those fed with cellulose, a decrease in the level of phospholipids was only observed in the rats that had been fed on guar gum or glucomannan. In addition, all WSDF feeding enhanced IgA productivity in the spleen and mesenteric lymph node lymphocytes, although the increase in serum IgA level was only observed in the rats fed on WSDF, and not on PHGG. When mesenteric lymph node lymphocytes were cultured in the presence of various concentrations of guar gum or glucomannan, no significant increase in Ig production was apparent. These data suggest that WSDF indirectly enhanced the Ig production of lymphocytes, and that serum lipid reduction and IgA production-enhancing activities of WSDF were dependent on their molecular sizes.

Effect of endoglucanase and cellobiohydrolase fromTrichoderma reeseion cellulose microfibril structure

Cellobiohydrolase (CBH, EC 3.2.91) was purified to homogeneity from Trichoderma reesei culture fluids by means of preparative isoelectric focussing (IEF). Its isoelectric points was 4.2. The degradation product of crystalline cellulose (Avicel and cotton) was predominantly cellobiose. The action of purified endoglucanase (EG) and CBH on cellulose microfibrils was followed by transmission electron microscopy (TEM) observations after Pt-C shadowing of the specimen. EG pretreatment of microfibrils resulted in submicrofibril formation. Addition of CBH induced the conversion of submicrofibrils into heterogeneous cellulose clusters and into homogeneous cellulose plaques. One structural effect of CBH was the increase in accessible cellulose surface area, possibly providing intermolecular entrace of water molecules between adjacent cellulose chains. Plaque formation is interpreted as a visible CBH action on crystalline cellulose to form swollen water-insoluble cellulose intermediates.

Effect of endoglucanase and cellobiohydrolase from Trichoderma reesei on cellulose microfibril structure

Cellobiohydrolase (CBH, EC 3.2.91) was purified to homogeneity from Trichoderma reesei culture fluids by means of preparative isoelectric focussing (IEF). Its isoelectric points was 4.2. The degradation product of crystalline cellulose (Avicel and cotton) was predominantly cellobiose. The action of purified endoglucanase (EG) and CBH on cellulose microfibrils was followed by transmission electron microscopy (TEM) observations after Pt-C shadowing of the specimen. EG pretreatment of microfibrils resulted in submicrofibril formation. Addition of CBH induced the conversion of submicrofibrils into heterogeneous cellulose clusters and into homogeneous cellulose plaques. One structural effect of CBH was the increase in accessible cellulose surface area, possibly providing intermolecular entrace of water molecules between adjacent cellulose chains. Plaque formation is interpreted as a visible CBH action on crystalline cellulose to form swollen water-insoluble cellulose intermediates.

Microcrystalline cellulose-hydrolyzing cellulase (endo-cellulase) from Trichoderma reesei CDU-11.

A microcrystalline cellulose (Avicel)-hydrolyzing cellulase (an endocellulase) was isolated from a preparation of Trichoderma reesei CDU-11 and purified successively by DEAE-Sepharose CL 6B and Concanavalin A-Sepharose column chromatography, and preparative electrophoresis and on Sephadex G-100 gel filtration. The cellulase preparation showed a single protein band on SDS-PAGE slab and gel disc electrophoresis. Its molecular weight was estimated to be 64, 000 by Sephadex G-100 gel filtration and 67, 000 by SDS slab electrophoresis. The isoelectric point was at pH 4.75 on polyacrylamide gel electric focusing. This cellulase hydrolyzed more specifically water insoluble microcrystalline cellulose such as Avicel rather than water soluble cellulose such as carboxymethyl-cellulose. It produced glucose, cellobiose and cellotriose from cellotetraose and cellohexaose, and it produced a significant amount of glucose and cellobiose from Avicel. Based on these results, it appears that the cellulase should be regarded as an endo-type cellulase, although it hydrolyzes Avicel relatively easier than CM-cellulose. We obtained no evidence that this cellulase is an exo-type one named cellobiohydrolase (CBH) in the systematic nomenclature.

Purification and properties of endo-(1→4)-β-d-glucanase from Ruminococcus albus

An enzyme active against O-(carboxymethyl)cellulose (CMC) was purified from a synthetic medium containing ball-milled cellulose wherein Ruminococcus albus had been cultivated for 70 h. After 570-fold purification, a homogeneous enzyme was obtained in a yield of 3%. The enzyme degraded CMC (molecular weight, 180,000; degree of substitution, 0.6) to a smaller polymer having a molecular weight of ∼20,000, and generated a small proportion of glucose, but negligible proportions of such cello-saccharides as cellobiose, cellotriose, cellotetraose, or cellopentaose. The fact that the enzyme could produce water-insoluble fragments was discovered by dissolving substrate and products in Cadoxen solution. No water-soluble cello-oligomers were detected by thin-layer chromatography after degradation of water-insoluble cellulose by the purified enzyme. Therefore, the enzyme was classified as an endo-(1→4)-β-d-glucanase.