Enzymatic Degradation Of Cellulose Research Articles

The mechanism of enzymatic cellulose degradation. Purification and some properties of two different 1,4beta-glucan glucanohydrolases from Trichoderma viride.

1. A low-molecular-weight and a high-molecular-weight 1,4beta-glucan glucanohydrolase (Cx enzyme) have been isolated from a commercial cellulase preparation derived from culture filtrates of the fungus Trichoderma viride. 2. The purification method for the isolation of the low-molecular-weight enzyme is a three-step procedure including chromatography on Bio-Gel P-10, chromatography on a dipolar adsorbent (arginine-Sepharose 6 B) and isoelectric focusing. 3. The starting material for the isolation of the high-molecular-weight enzyme was pre-fractionated by chromatography on Bio-Gel P-10, by DEAE-Sephadex chromatography and by SE-Sephadex chromatography as described previously by us. Further fractionation of this material was achieved by affinity chromatography and repeated isoelectric focusing. 4. Free zone electrophoresis of the low-molecular-weight enzyme indicated a homogeneous protein. The high-molecular-weight enzyme was homogenous in sedimentation equilibrium analysis. 5. The molecular weights of the enzymes were 12 500 and 50 000 +/- 2000 respectively. The former value was determined by chromatography on a calibrated column of Bio-Gel P-100 and the latter value by sedimentation equilibrium analysis. 6. The low-molecular-weight enzyme was isoelectric at pH 4.60 (10 degrees C) and contained 21% carbohydrate. The corresponding values for the high-molecular-weight enzyme were pH 3.39 and 12%. 7. Both enzymes were active in releasing free fibers from filter-paper. The low-molecular-weight enzyme was estimated to be about twice as effective as the high-molecular-weight enzyme in this regard.

The Mechanism of Enzymatic Cellulose Degradation. Characterization and Enzymatic Properties of a beta-1,4-Glucan Cellobiohydrolase from Trichoderma viride

The Mechanism of Enzymatic Cellulose Degradation. Characterization and Enzymatic Properties of a beta-1,4-Glucan Cellobiohydrolase from Trichoderma viride

The mechanism of enzymatic cellulose degradation. Isolation and some properties of a beta-glucosidase from Trichoderma viride.

1. A β‐glucosidase (cellobiase) has been isolated from a commercial cellulase preparation derived from culture filtrates of the fungus Trichoderma viride.2. The crude material was pre‐fractionated by chromatography on Bio‐Gel P‐10 and by DEAE‐Sephadex chromatography as previously described [1]. Further fractionation methods utilized in the present paper included ammonium sulfate precipitation, SE‐Sephadex chromatography, isoelectiic focusing, and chromatography on Bio‐Gel P‐60.3. A yield of 53 mg enzyme was obtained per 100 g commercial cellulase. The specific activity of the purified enzyme represented an approximately 60‐fold increase from that of the commercial product.4. The isolated enzyme was homogeneous in polyacrylamide gel electrophoresis at pH 8.0 by isoelectric focusing in a polyacrylamide gel and also in free zone electrophoresis.5. No enzyme activity towards Avicel (microcrystalline cellulose) or towards carboxymethyl‐cellulose could be detected in the purified material under the assay conditions used.6. A molecular weight of 47000 was determined for the enzyme by dodecylsulfate‐polyacrylamide gel electrophoresis. The same molecular weight value was obtained by chromatography of the native enzyme on Bio‐Gel P‐100 and also by chromatography of the reduced and alkylated enzyme on Sepharose 6B in 6 M guanidine‐HCl.7. The amino‐acid analysis showed that the enzyme was rich in aromatic (9.5%) and acidic (19.0%) amino acids. The half‐cystine content was 6 residues per molecule indicating 3 disulfide bridges, since no free sulfhydryl group was detectable. The purified enzyme contained no carbohydrate (as hexose).8. The enzyme was isoelectric at pH 5.74 (10 °C) and the electrophoretic mobility was determined to be 1.49 × 10−5 cm2 s−l V− at pH 8.0.9. The Km‐values for the enzyme towards the substrates p‐ nitrophenyl‐β‐d‐glucoside, cellobiose, and cellotetraose were found to be 0.28, 1.5, and 0.35 mM, respectively.

The Mechanism of Enzymatic Cellulose Degradation

A cellulolytic enzyme (“C1” enzyme) has been isolated from a commercial cellulase preparation derived from culture filtrates of the fungus Trichoderma viride. The purification method is a four‐step procedure including chromatography on Bio‐Gel P‐10, DEAE‐Sephadex chromatography, isoelectric focusing and chromatography on Bio‐Gel P‐60. A yield of 144 mg enzyme was obtained per 100 g commercial cellulase. The isolated enzyme was homogeneous in polyacrylamide gel electrophoresis at pH 5.0 and at pH 8.0 by isoelectric focusing in a polyacrylamide gel and also in the ultracentrifuge. No enzyme activity towards carboxymethylcellulose could be detected in the purified material under the assay conditions used. Similarly, there was no β‐glucosidase activity. The purified enzyme was associated with 3.3% carbohydrate and is assumed to be a glycoprotein. The enzyme was isoelectric at pH 3.79 (10°C). A molecular weight of 46000 was determined by chromatography of the reduced and alkylated enzyme on a calibrated column of Sepharose 6B in 6 M guanidine‐HCl. Crystalline cellulose (Avicel), phosphoric acid‐swollen Avicel and cellotetraose were degraded by the enzyme and in each case the principle reaction product was cellobiose. Evidence indicates that the purified enzyme is a β‐1,4‐glucan cellobiohydrolase.

Enzymatic degradation of cellulose, cellulose derivatives and hemicelluloses in relation to the fungal decay of wood

Cellulases and hemicellulases (mannanase and xylanase) from culture filtrates of various fungi, including a brown rot fungus, Polyporus schweinitzii and a soft rot fungus, Chaetomium globosum, have been isolated and characterized. Investigations of their molecular weights, pH- and temperature optima and stabilities indicate that hydrolases of the wood destroying fungi are very similar to one another and to hydrolases from various other plant organisms. Substrate specificities and the effect of substituents on the natural substrates have also been investigated. Routine tests for mannanase and xylanase were carried out with model substrates (mannan from Tubera salep, xylan from wheat straw), but preliminary tests with complex hemicelluloses isolated from wood showed that these too could be broken down to monoor oligosaccharides. The results presented are discussed in relation to possible roles of the various enzymes in vivo.

The enzymatic degradation of cellulose from Valonia ventricosa

The degradation of microfibrils from Valonia ventricosa by cellulase has been studied. As a result of enzymatic attack the elementary fibrils making up the microfibrils tended to separate and the ends of the microfibrils became oblique or pointed. The terminal planes made angles of 60 ... 66°, 33°, or 20 ... 25° with the microfibril axis. These planes are assumed to correspond to the 41\(\bar 4\), 43\(\bar 4\) and 45\(\bar 4\) planes of the cellulose lattice and it is suggested that they are planes along which it is progressively more difficult for hydrolysis to proceed. On the basis of these considerations a suggestion has been proposed to explain the form of erosion cavities formed by soft-rot fungi described by previous workers in wood fibres and tracheids.

Studies of the Enzymatic Degradation of Cellulose

Studies of the Enzymatic Degradation of Cellulose