Thomas M. Wood

Purification and Some Properties of the Extracellular β-d-Glucosidase of the Cellulolytic Fungus Trichoderma koningii

SUMMARY: The β-d-glucosidase which was associated with the cellobiohydrolase and endo-(1·4)-β-d-glucanase activities in the cellulase of the fungus Trichoderma koningii was purified by gel filtration on a column of Ultrogel AcA 44, ion-exchange chromatography on DEAE-Sepharose and sulphoethyl-Sephadex (SE-Sephadex), and finally by isoelectric focusing in a pH gradient supported in a sucrose density gradient. The separation of β-glucosidase and endo-(l·4)-β-glucanase, which was effected with some difficulty on SE-Sephadex, could be achieved readily by chromatography on a column of concanavalin A-Sepharose. Isoelectric focusing yielded two β-glucosidase components (pI 5·53 and 5·85) of identical molecular weight (39800), but with different affinities for o-nitrophenyl-β-glucoside (K m 0·37 · 0·04 and 0·85 · 0·3 mm), cellobiose (K m 1·18 · 0·09 and 0·86 · 0·02mm) cellotriose (K m 5·10 · 0·22 and 7·10 · 0·33 mm), cellotetraose (K m 2·38 · 0·14 and 4·0 · 0·4mm) and cellopentaose (K m 1·51 · 0·04 and 2·19 · 0·06 mm). One of the β-glucosidases was devoid of carbohydrate, the other contained approximately 2% carbohydrate. Gluconolactone was a powerful inhibitor of the action of both enzymes on o-nitrophenyl-β-glucoside (K i 1·8 · 0·08 and 1·17 · 0·05 μm); glucose was less effective (K i 1·05 · 0·07 and 0·66 · 0·04 mm). Inhibition was of the competitive type in each case. Both β-glucosidases showed the same capacity for acting in synergism with a mixture of the endo-(l·4)-β-glucanase and cellobiohydrolase in solubilizing the cellulose in cotton fibre. They differed markedly, however, in the degree of synergism they showed when acting in concert with the cellobiohydrolase on partially degraded H3PO4-swollen cellulose.

Purification and characterization of three endo-(1,4)-β-xylanases and one β-xylosidase from Aspergillus awamori

Abstract Three endo-(1,4)-β-xylanases (endo-xylanase I, II, and III) and one β- d -xyloside xylohydrolase (β-xylosidase) were purified from a crude culture filtrate of Aspergillus awamori CMI 142717, grown on milled oat straw as carbon source. Aspergillus awamori xylanases differ in some characteristics of known xylanases. The optimum pH for the endo-xylanases were between 4.0 and 5.5 and the optimum temperature between 45°C and 55°C; β-xylosidase was optimal around pH 6.5 and 70°C. All endo-xylanases were able to degrade xylan to xylobiose and xylotriose. Endo-xylanase I also produced small amounts of xylose. The molecular weights of endo-xylanase I, II, and III were, respectively, 39 000, 23 000, and 26 000. The molecular weight of β-xylosidase was 110 000. The specific activities of endo-xylanase I, II, and III towards water-soluble oat spelts arabinoxylan were, respectively, 69.6 U mg −1 , 68.6 U mg −1 , and 16.3 U mg −1 . The specific activity of β-xylosidase towards p -nitrophenyl-β-xylopyranoside was 34.1 U mg −1 . The activity of these enzymes was significantly inhibited by Hg 2+ , Pb 2+ , and Ag + .

Purification and characterization of a (1,4)-ß-D-arabinoxylan arabinofuranohydrolase from Aspergillus awamori.

SummaryAn enzyme able to split off arabinose sidechains from cereal arabinoxylans was isolated from a cell-free culture filtrate of Aspergillus awamori CMI 142717 containing milled oat straw as the carbon source. The enzyme was highly specific for arabinoxylans and, unlike other α-l-arabinofuranosidases reported in the literature, did not show any activity towards p-nitrophenyl α-l-arabinofuranoside, arabinans and arabinogalactans. This novel enzyme, which can be described as a (1,4)-β-d-arabinofuranohydrolase, had a molecular mass of 32 000 Da when determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and a specific activity of 22 units/mg on wheat arabinoxylan.

The anaerobic fungus Neocallimastix frontalis: isolation and properties of a cellulosome-type enzyme fraction with the capacity to solubilize hydrogen-bond-ordered cellulose

SummaryA minor component isolated from the extra-cellular cellulase of the anaerobic rumen fungus Neocallimastix frontalis by adsorption on cellulose had a remarkable capacity to degrade crystalline hydrogen-bond-ordered cellulose. When produced in a semi-defined medium the component comprised normally less than 4% of the total protein and only 0.3% of the protein in cultures containing rumen fluid. The minor component showed endoglucanase (carboxymethylcellulase) and β-glucosidase activity and effected the extensive hydrolysis of “crystalline” cellulose in the form of the cotton fibre when acting alone. Glucose was the sole product of hydrolysis. The specific activity of the crystalline cellulose solubilizing factor (CCSF) in degrading cotton fibre was much higher than any other cellulase or cellulase component reported so far. The activity of the CSSF to crystalline hydrogen-bond-ordered cellulose resides in a high molecular mass complex of 670 kDa, that comprised a number of subunits ranging in size from 68 to 135 kDa.

Purification and properties of a feruloyl/ϱ-coumaroyl esterase from the fungus Penicillium pinophilum

A purified extracellular phenolic acid esterase, produced by the fungus Penicillium pinophilum in solid-state culture, released ferulic and ϱ-coumaric acid from methyl esters of the acids. The esterase was also able to release all the alkali-extractable feruloyl and 80% of the ϱ-coumaroyl groups from a water-soluble wheat straw xylan without degrading the polysaccharide, but it showed no capacity to release ferulic and ϱ-coumaric acid from a grass cell wall preparation in the absence of other xylanolytic enzymes. Synergistic effects were observed between the esterase and other xylan-degrading enzymes in hydrolyzing the ferulic and, to a lesser extent, the ϱ-coumaric acid esters from both wheat straw xylan and the grass cell walls. The esterase had an apparent molecular mass of 57 kDa by SDS-PAGE and an isoelectric pH of 4.6: temperature and pH optima were 55°C and 6.0, respectively. Cu2+ and Fe2+ were inhibitory, but Zn2+, Ca2+, Ag+, and Mg2+ stimulated activity. The enzyme had a higher affinity towards the methyl ester of ϱ-coumaric acid than towards the methyl ester of ferulic acid: the apparent Km values for the ferulic acid and ϱ-coumaric acid esters were 0.14 and 0.08 mM, respectively; the Vmax values were 32.5 and 37.9 μmol min-1 mg-1, respectively.

Xylan-degrading enzyme system produced by the fungus Aspergillus awamori : Isolation and characterization of a feruloyl esterase and a p-coumaroyl esterase

Abstract Feruloyl esterase and p -coumaroyl esterase activities were separated by ion-exchange chromatography on a column of DEAE-Sepharose CL 6B and then purified by hydrophobic interaction chromatography and anion-exchange chromatography, respectively. Feruloyl esterase had an apparent molecular mass of 112 kDa (SDS-PAGE) and an isoelectric pH of 3.7; the p -coumaroyl esterase was much smaller (75 kDa) and had a pI of 4.2. The feruloyl esterase was highly specific for the methyl ester of ferulic acid and for feruloyl esters associated with a water-soluble xylan polysaccharide from wheat straw; p -coumaroyl esterase was also highly specific for the corresponding p -coumaroyl ester substrates. The wheat straw xylan was de-esterified by the esterases without prior degradation of the polysaccharide; however, esterified short-chain xylooligosaccharides which were generated by a purified fungal xylanase were better substrates. The apparent K m values for the feruloyl and p -coumaroyl residues were 0.93 ± 0.05 and 0.031 ± 0.02 mM, respectively, using the methyl ester substrates; the respective V max values were 2.82 ± 0.05 and 0.57 ± 0.03 μ mol min −1 mg −1 protein. The esterases had only a small capacity to release ferulic and p -coumaric acids from cell walls of various plants, including mesophyll cell walls from rye grass, but they were more effective when acting in concert with the commercially available plant cell wall-degrading enzyme Celluclast.

Cellulase from Fusarium solani: purification and properties of the C1 component.

The C1 component from Fusarium solani cellulase was purified extensively by molecular-sieve chromatography on Ultrogel AcA-54 and ion-exchange chromatography on DEAE-Sephadex. The purified component showed little capacity for hydrolysing highly ordered substrates (e.g., cotton fibre), but poorly ordered substrates (e.g., H3PO4-swollen cellulose), and the soluble cello-oligosaccharides cellotetraose and cellohexaose, were readily hydrolysed; cellobiose was the principal product in each case. Attack on O(-carboxymethyl)cellulose, a substrate widely used for measuring the activity of the randomly acting enzymes (Cx enzymes) of the cellulase complex, was minimal, and ceased after the removal of a few unsubstituted residues from the end of the chain. These observations, and the fact that the rate of change of degree of polymerisation of H3PO4-swollen cellulose was very slow compared with that effected by the randomly acting endoglucanases (Cx, CM-cellulases), indicate that C1 is a cellobiohydrolase. Fractionation by a variety of methods gave no evidence for the non-identity of the cellobiohydrolase and the component that acted in synergism with the randomly acting Cx enzyme when solubilizing cotton fibre.

Studies on the cellulase of the rumen anaerobic fungus Neocallimastix frontalis, with special reference to the capacity of the enzyme to degrade crystalline cellulose

Abstract By using cotton fiber, carboxymethylcellulose (CM-cellulose), and o -nitrophenyl-β- d -glucoside as substrates, it was possible to demonstrate that there were at least three different types of enzyme present in culture filtrates of Neocallimastix frontalis RK21. The activity to crystalline cellulose (cotton fiber) resided in a high-molecular-weight complex that comprised endoglucanase activity, β-glucosidase activity, and another enzyme. However, synergism between the components in the high-molecular-weight complex and between the complex and low-molecular-weight endoglucanases and β-glucosidases was also apparent in the solubilization of crystalline cellulose. The composition of the complex varied according to the growth conditions: it was, however, between 750 and 1000 kDa in size. Cultures containing rumen fluid contained only small amounts of the high-molecular-weight complex. Cultures grown on defined medium were rich in high-molecular-weight complex, but only when the concentration of the carbon source was less than 1.0%. Treatment of the crude culture filtrates with chitinase under conditions that had little effect on the activity of the enzyme to endoglucanase (CM-cellulose) or β-glucosidase completely destroyed the activity to crystalline cellulose. It is tentatively suggested that an enzyme crucial for the activity to crystalline cellulose may be cell wall-bound and may be dependent on its association with the cell wall for the maintenance of its conformation for attacking crystalline cellulose. The enzyme involved in degrading crystalline cellulose is much more thermolabile than the CM-cellulase or the β-glucosidase: activity is optimal at pH 6.0 and 40°C.

Purification and some properties of a (1→4)-β-d-glucan glucohydrolase associated with the cellulase from the fungus Penicillium funiculosum

Abstract The (1→4)-β- d -glucan glucohydrolase from Penicillium funiculosum cellulase was purified to homogeneity by chromatography on DEAE-Sephadex and by iso-electric focusing. The purified component, which had a molecular weight of 65,000 and a pI of 4.65, showed activity on H 3 PO 4 -swollen cellulose, o -nitrophenyl β- d -glucopyranoside, cellobiose, cellotriose, cellotetraose, and cellopentaose, the K m values being 172 mg/mL, and 0.77, 10.0, 0.44, 0.77, and 0.37 m m , respectively. d -Glucono-1,5-lactone was a powerful inhibitor of the action of the enzyme on o -nitrophenyl β- d -glucopyranoside ( K i 2.1 μ m ), cellobiose ( K i 1.95 μ m ), and cellotriose ( K i 7.9 μ m ) [ cf. d -glucose ( K i 1756 μ m )]. On the basis of a Dixon plot, the hydrolysis of o -nitrophenyl β- d -glucopyranoside appeared to be competitively inhibited by d -glucono-1,5-lactone. However, inhibition of hydrolysis by d -glucose was non-competitive, as was that for the gluconolactone-cellobiose and gluconolactone-cellotriose systems. Sophorose, laminaribiose, and gentiobiose were attacked at different rates, but the action on soluble O -(carboxymethyl)cellulose was minimal. The enzyme did not act in synergism with the endo-(1→4)-β- d -glucanase component to solubilise highly ordered cotton cellulose, a behaviour which contrasts with that of the other exo-(1→4)-β- d -glucanase found in the same cellulase, namely, the (1→4)-β- d -glucan cellobiohydrolase.

Studies of two low-molecular-weight endo-1(1→4)-β-d-xylanases constitutively synthesised by the cellulolytic fungus Trichoderma koningii

Abstract Two endo-(1→4)-β- d -xylanases (xylanases 1 and 2), which were constitutively synthesised by the fungus Trichoderma koningii , were purified to homogeneity on gel-filtration media and by isoelectric focusing. They had molecular weights of 29,000 (xylanase 1) and 18,000 (xylanase 2), and isoelectric pHs of 7.24 (xylanase 1) and 7.3 (xylanase 2); neither enzyme was associated with carbohydrate. Xylanase 1 had an optimum at the remarkably high temperature of 60–65°. Each enzyme liberated a different range of oligosaccharides from oat-straw arabinoxylan, but only xylanase 1 released l -arabinose and d -xylose. Both xylanases were free from cellulase activity.