EndB, a multidomain family 44 cellulase from Ruminococcus flavefaciens 17, binds to cellulose via a novel cellulose-binding module and to another R. flavefaciens protein via a dockerin domain.

The mechanisms by which cellulolytic enzymes and enzyme complexes in Ruminococcus spp. bind to cellulose are not fully understood. The product of the newly isolated cellulase gene endB from Ruminococcus flavefaciens 17 was purified as a His-tagged product after expression in Escherichia coli and found to be able to bind directly to crystalline cellulose. The ability to bind cellulose is shown to be associated with a novel cellulose-binding module (CBM) located within a region of 200 amino acids that is unrelated to known protein sequences. EndB (808 amino acids) also contains a catalytic domain belonging to glycoside hydrolase family 44 and a C-terminal dockerin-like domain. Purified EndB is also shown to bind specifically via its dockerin domain to a polypeptide of ca. 130 kDa present among supernatant proteins from Avicel-grown R. flavefaciens that attach to cellulose. The protein to which EndB attaches is a strong candidate for the scaffolding component of a cellulosome-like multienzyme complex recently identified in this species (S.-Y. Ding et al., J. Bacteriol. 183:1945-1953, 2001). It is concluded that binding of EndB to cellulose may occur both through its own CBM and potentially also through its involvement in a cellulosome complex.

Cellulosomal scaffoldin-like proteins from Ruminococcus flavefaciens.

Two tandem cellulosome-associated genes were identified in the cellulolytic rumen bacterium, Ruminococcus flavefaciens. The deduced gene products represent multimodular scaffoldin-related proteins (termed ScaA and ScaB), both of which include several copies of explicit cellulosome signature sequences. The scaB gene was completely sequenced, and its upstream neighbor scaA was partially sequenced. The sequenced portion of scaA contains repeating cohesin modules and a C-terminal dockerin domain. ScaB contains seven relatively divergent cohesin modules, two extremely long T-rich linkers, and a C-terminal domain of unknown function. Collectively, the cohesins of ScaA and ScaB are phylogenetically distinct from the previously described type I and type II cohesins, and we propose that they define a new group, which we designated here type III cohesins. Selected modules from both genes were overexpressed in Escherichia coli, and the recombinant proteins were used as probes in affinity-blotting experiments. The results strongly indicate that ScaA serves as a cellulosomal scaffoldin-like protein for several R. flavefaciens enzymes. The data are supported by the direct interaction of a recombinant ScaA cohesin with an expressed dockerin-containing enzyme construct from the same bacterium. The evidence also demonstrates that the ScaA dockerin binds to a specialized cohesin(s) on ScaB, suggesting that ScaB may act as an anchoring protein, linked either directly or indirectly to the bacterial cell surface. This study is the first direct demonstration in a cellulolytic rumen bacterium of a cellulosome system, mediated by distinctive cohesin-dockerin interactions.

Isolation and overexpression of a gene encoding an extracellular beta-(1,3-1,4)-glucanase from Streptococcus bovis JB1.

Streptococcus bovis JB1 was found to produce a 25-kDa extracellular enzyme active against beta-(1,3-1,4)-glucans. A gene was isolated encoding a specific beta-(1,3-1,4)-glucanase that corresponds to this size and belongs to glycoside hydrolase family 16. A 4- to 10-fold increase in supernatant beta-glucanase activity was obtained when the cloned beta-glucanase gene was reintroduced into S. bovis JB1 by use of constructs based on the plasmid vector pTRW10 or pIL253. The beta-(1,3-1,4)-glucanase gene was also expressed upon introduction of the pTRW10 construct pTRWL1R into Lactococcus lactis IL2661 and Enterococcus faecalis JH2-SS, although extracellular activity was 8- to 50-fold lower than that in S. bovis JB1. The beta-(1,3-1,4)-glucanase purified from the culture supernatant of S. bovis JB1 carrying pTRWL1R showed a K(m) of 2.8 mg per ml and a Vmax of 338 mumol of glucose equivalents per min per mg of protein with barley beta-glucan as the substrate. The S. bovis beta-(1,3-1,4)-glucanase may contribute to the ability of this bacterium to utilize starch by degrading structural polysaccharides present in endosperm cell walls.

Arabinoxylan-degrading enzyme system of the fungus Aspergillus awamori: purification and properties of an alpha-L-arabinofuranosidase.

An alpha-L-arabinofuranosidase produced by the fungus Aspergillus awamori had a molecular mass of approximately 64 kDa on sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE) and was optimally active at pH 4.6 and 50 degrees C. The enzyme, which chromatographed as a single component on SDS-PAGE, appeared to consist of two isoenzymes of pI 3.6 and 3.2. Acting in isolation, the alpha-L-arabinofuranosidase had only a very limited capacity to release L-arabinose (less than 11%) directly from arabinoxylans that had been extracted from a number of plant cell wall preparations using 18% alkali, but a much higher proportion of the L-arabinose (46%) was released from a wheat straw arabinoxylan that had been isolated by steam treatment. There was a marked synergistic effect between the alpha-L-arabinofuranosidase and an endo-(1 --> 4)-beta-D-xylanase produced by A. awamori in both the rate and extent of the release of L-arabinose from both oat straw and wheat straw arabinoxylans, suggesting that L-arabinose-substituted oligosaccharides generated by the endoxylanase action were better substrates for enzyme action. A novel property of the alpha-L-arabinofuranosidase was its capacity to release a substantial proportion (42%) of feruloyl L-arabinose from intact wheat straw arabinoxylan. The concerted action of the alpha-L-arabinofuranosidase and endoxylanase released 71% of the feruloyl L-arabinose and 69% of the p-coumaroyl L-arabinose substituents from wheat straw arabinoxylan.

The cellulase system of the anaerobic rumen fungus Neocallimastix frontalis: studies on the properties of fractions rich in endo-(1-->4)-beta-D-glucanase activity.

Seven fractions rich in endoglucanase activity were separated from the extracellular cellulase system of the anaerobic rumen fungus Neocallimastix frontalis. The fractions (ES1, ES3, ES2U1, ES2U2, ES2U4, ES2U3C1 and ES2U3C2) were separated from each other and from a fraction that could solubilize crystalline cellulose (the so-called crystalline-cellulose-solubilizing component, CCSC) by the sequential use of differential adsorption on the microcrystalline cellulose Avicel, gel filtration and affinity chromatography on concanavalin-A-Sepharose. The molecular masses of the endoglucanase fractions, when determined by gel filtration, were 64, 30, 61, 113, 17, 38 and 93 kDa respectively. Each enzyme degraded carboxymethylcellulose and was rich in activity to cellulose swollen in phosphoric acid to break the hydrogen bonding: cellobiose, cellotriose and cellotetraose were released in differing proportions. Each fraction showed a characteristic gradient when the capacity of each enzyme to increase the fluidity of a solution of carboxymethylcellulose was plotted against the increase in reducing power of the solution. Although neither endoglucanase fraction, acting in isolation, could degrade crystalline cellulose, three of the fractions (ES1, ES3 and ES2U1) could act synergistically with the CCSC fraction in this regard. Remarkably, the same three fractions also acted in synergism with the cellobiohydrolases (CBH I and CBH II) of the aerobic fungus Penicillium pinophilum in degrading crystalline cellulose, but only when both cellobiohydrolase enzymes were present in the solution along with any one of the three endoglucanases. These observations support the conclusion that the mechanism of action of the cellulase system of N. frontalis in degrading crystalline cellulose may be similar to that operating in the aerobic fungi.

D-xylan-degrading enzyme system from the fungus Phanerochaete chrysosporium: isolation and partial characterisation of an alpha-(4-O-methyl)-D-glucuronidase.

A number of fungi were screened for their capacities to produce extracellular alpha-(4-O-methyl)-D-glucuronidase. Of those tested, Phanerochaete chrysosporium ATCC 24725 produced the enzyme in greatest yield. The single alpha-(4-O-methyl)-D-glucuronidase produced by this fungus was purified by a series of chromatographic methods involving anion exchange, hydrophobic interaction and chromatofocusing. Isolated in this way, the enzyme had an apparent molecular mass of 112 kDa in sodium dodecyl sulphate polyacrylamide gels, and a pI of 4.6 when determined by isoelectric focusing in polyacrylamide gels. The enzyme was optimally active at pH 3.5, but showed significant activity over the pH range 3-5. In the absence of substrate the enzyme was inactivated at pH 3.5 in 2 h at 50 degree C: at pH 5.0 it retained 42% of its activity for 24 h at this temperature. The enzyme showed little activity on glucuronoxylan polysaccharides, but some short-chain xylo-oligosaccharides which were substituted with alpha-linked 4-O-methyl-D-glucopyranosyl uronic acid attached to the 2-position of the non-reducing D-xylopyranosyl residue were readily hydrolysed. There were marked synergistic effects apparent in the release of 4-O-methyl-D-glucopyranosyl uronic acid from various glucuronoxylans when the alpha-(4-O-methyl)-D-glucuronidase was acting in concert with endo-(1-->4)-beta-D-xylanase, and with beta-D-xylosidase and/or an alpha-L-arabinofuranosidase.

Hydrolysis of oligosaccharides of the ß-(1→4)-linked D-xylose series by an endo(1→4)-ß-D-xylanase from the anaerobic rumen fungus Neocallimastix frontalis.

An endo-(1→4)-ß-D-xylanase from Neocallimastix frontalis was purified by anion-exchange chromatography. The enzyme had an apparent molecular mass of 30 kDa on SDS-PAGE and exhibited maximum activity at 50°C and at pH values between 6.0 and 6.6. Kinetic studies on the hydrolysis of xylo-oligosaccharides, ranging from xylobiose to xylodecaose, showed that xylohexaose and xyloheptaose were the preferred substrates for the enzyme and that xylobiose, xylotriose and xylotetraose were not hydrolysed. Xylose was not a product of the hydrolysis of any of the xylo-oligosaccharide substrates tested. The enzyme appeared to have a strong preference for the hydrolysis of the internal glycosidic bonds of the oligosaccharides, which is typical of endo-(1→4)-ß-D-xylanase activity, but it differed from other fungal endo-(1→4)-ß-D-xylanases in that it had uniform action on the various internal linkages in the xylo-oligosaccharides.

Mode of action, kinetic properties and physicochemical characterization of two different domains of a bifunctional (1-->4)-beta-D-xylanase from Ruminococcus flavefaciens expressed separately in Escherichia coli.

Two catalytic domains, A and C, of xylanase A (XYLA) from Ruminococcus flavefaciens were expressed separately as truncated gene products from lacZ fusions in Escherichia coli. The fusion products, referred to respectively as XYLA-A1 and XYLA-C2, were purified to homogeneity by anion-exchange chromatography and chromatofocusing. XYLA-A1 was isoelectric at pH 5.0 and had a molecular mass of 30 kDa, whereas XYLA-C2 had a pI of 5.4 and a molecular mass of 44 kDa. The catalytic activity shown by both domains was optimal at 50 degrees C, but XYLA-A1 was more sensitive than XYLA-C2 to temperatures higher than the optimum. XYLA-A1 showed a higher sensitivity to pH than XYLA-C2. The enzyme activity of both domains was completely inactivated in the presence of copper or silver ions and partially inactivated by iron or zinc ions. Neither domain was active on xylo-oligosaccharides shorter than xylopentaose: the rate of degradation of longer xylo-oligosaccharides (degree of polymerization 5-10) increased as the chain length increased. Analysis of the products of hydrolysis of xylo-oligosaccharides and xylan (arabinoxylan) polysaccharide showed that the two domains differed in their modes of action: xylobiose was the shortest product of the hydrolysis. With oat spelt xylan as substrate, XYLA-A1 activity was apparently restricted to regions where xylopyranosyl residues did not carry arabinofuranosyl substituents, whereas XYLA-C2 was able to release hetero-oligosaccharides carrying arabinofuranosyl residues. Neither domain was able to release arabinose from oat spelt xylan.

The mechanism of fungal cellulase action. Synergism between enzyme components of Penicillium pinophilum cellulase in solubilizing hydrogen bond-ordered cellulose.

Studies on reconstituted mixtures of extensively purified cellobiohydrolases I and II and the five major endoglucanases of the fungus Penicillium pinophilum have provided some new information on the mechanism by which crystalline cellulose in the form of the cotton fibre is rendered soluble. It was observed that there was little or no synergistic activity either between purified cellobiohydrolases I and II, or, contrary to previous findings, between the individual cellobiohydrolases and the endoglucanases. Cotton fibre was degraded to a significant degree only when three enzymes were present in the reconstituted enzyme mixture: these were cellobiohydrolases I and II and some specific endoglucanases. The optimum ratio of the cellobiohydrolases was 1:1. Only a trace of endoglucanase activity was required to make the mixture of cellobiohydrolases I and II effective. The addition of cellobiohydrolases I and II individually to endoglucanases from other cellulolytic fungi resulted in little synergistic activity; however, a mixture of endoglucanases and both cellobiohydrolases was effective. It is suggested that current concepts of the mechanism of cellulase action may be the result of incompletely resolved complexes between cellobiohydrolase and endoglucanase activities. It was found that such complexes in filtrates of P. pinophilium or Trichoderma reesei were easily resolved using affinity chromatography on a column of p-aminobenzyl-1-thio-beta-D-cellobioside.

Hydrolysis of the polysaccharides of straw by enzymes produced by a mutant strain of the fungus Penicillium pinophilum.

The saccharification of the polysaccharides of barley, oat, and wheat straws and Solka Floc was studied using the extracellular enzyme system synthesized by mutant strain NTG III/6 of the fungus Penicillium pinophilum 87160iii. The enzymes obtained in cultures containing Solka Floc or barley straw as the carbon source were compared. Solka Floc at 10% (w/v) concentration was hydrolyzed to the extent of 70% in 72 h at 50 degrees C using a reaction mixture containing 7 filter paper units/mL of cellulase induced on Solka Floc, but hydrolysis was increased to 90% when the enzyme induced on barley straw was used. Under the same conditions, the polysaccharides in barley, oat, and wheat straws were hydrolyzed, respectively, in 72 h, to the extent of 42-48%, 62%, and 52%, but hydrolysis was increased to 93%, 100%, and 92%, respectively, after treatment of the substrates with alkaline-H(2)O(2) reagent at room temperature.

The cellulase of Penicillium pinophilum. Synergism between enzyme components in solubilizing cellulose with special reference to the involvement of two immunologically distinct cellobiohydrolases.

Two immunologically unrelated cellobiohydrolases (I and II), isolated from the extracellular cellulase system elaborated by the fungus Penicillum pinophilum, acted in synergism to solubilize the microcrystalline cellulose Avicel; the ratio of the two enzymes for maximum rate of attack was approx. 1:1. A hypothesis to explain the phenomenon of synergism between two endwise-acting cellobiohydrolases is presented. It is suggested that the cellobiohydrolases may be two stereospecific enzymes concerned with the hydrolysis of the two different configurations of non-reducing end groups that would exist in cellulose. Only one type of cellobiohydrolase has been isolated so far from the cellulases of the fungi Fusarium solani and Trichoderma koningii. Only cellobiohydrolase II of P. pinophilum acted synergistically with the cellobiohydrolase of the fungi T. koningii or F. solani to solubilize Avicel. Cellobiohydrolase II showed no capacity for co-operating with the endo-1,4-beta-glucanase of T. koningii or F. solani to solubilize crystalline cellulose, but cellobiohydrolase I did. These results are discussed in the context of the hypothesis presented.

Isolation and characterization of the 1,4-beta-D-glucan glucanohydrolases of Talaromyces emersonii.

Culture filtrates of Talaromyces emersonii were found to contain four endocellulases termed I, II, III and IV, the last having the greatest electrophoretic mobility towards the anode in homogeneous 5%-(w/v)-polyacrylamide gels at pH 4.5. All four are glycoproteins, the carbohydrate contents being: I, 27.7%; II, 29.0%; III, 44.7%; IV, 50.8. Each form is eluted as a single peak corresponding to an Mr value of 68000 on gel filtration at pH 3.5 and as a single band corresponding to an Mr value of 35000 on reductive sodium dodecyl sulphate/polyacrylamide-gradient-gel electrophoresis. However, we believe that the latter represents the native Mr value. The pI values for each lie between pH 2.8 and 3.2. Activity in each case is optimal at pH 5.5-5.8 and at 75-80 degrees C. Half-life values at pH5 and 75 degrees C were from 2 to 4h. The specific activity with any individual substrate was much the same for each enzyme, as was the ratio of activity from one substrate to the next. Possible reasons for the observation that plots of velocity versus substrate concentration are sigmoidal are discussed. We believe that the finding of four endocellulases reflects differential glycosylation of a single enzyme form rather than genetically determined differences in primary structure.

The isolation, purification and properties of the cellobiohydrolase component of Penicillium funiculosum cellulase.

1. A cellobiohydrolase component was isolated from a Penicillium funiculosum cellulase preparation by chromatography on DEAE-Sephadex, and purified by isoelectric focusing. 2. Purified in this way, the enzyme was homogeneous as judged by electrophoresis on sodium dodecyl sulphate/polyacrylamide gels and isoelectric focusing in polyacrylamide gels. 3. Acting in isolation, the enzyme had little hydrolytic activity to highly ordered celluloses such as cotton fibre, but, when recombined in the original proportions with the other components [endo-(1 leads to 4)-beta-D-glucanase and beta-D-glucosidase] of the complex, 98% of the original activity was recovered. 4. Synergistic effects were also observed when the enzyme was acting in concert with endo-(1 leads to 4)-beta-D-glucanase from other fungal sources. 5. Less-well-ordered celluloses, such as that swollen in H3PO4, were extensively hydrolysed, the principal product being cellobiose. 6. Attack on carboxymethyl-cellulose (CM-cellulose), which is the substrate normally used to assay for endo-(1 leads to 4)-beta-D-glucanase activity, was minimal. 7. The enzyme was associated with 9% of neutral sugar, 88% of which was mannose. It was isoelectric at pH 4.36 (4 degrees C) and had a mol.wt. of 46 300 (determined by gel chromatography on a calibrated column of Ultrogel). 8. The enzyme was specific for the beta-(1 leads to 4)-linkage.

The cellulase of Trichoderma koningii. Purification and properties of some endoglucanase components with special reference to their action on cellulose when acting alone and in synergism with the cellobiohydrolase.

1. Four principal endoglucanase components of Trichoderma koningii cellulase were separated and purified by gel filtration on Sephadex G-75, ion-exchange chromatography on DEAE- and sulphoethyl-Sephadex and isoelectric focusing. 2. All four endoglucanases hydrolysed CM-cellulose, H3PO4-swollen cellulose, cellotetraose and cellopentaose, but differed in the rate and mode of attack. 3. Attack on cotton fibre by the endoglucanases was minimal, but resulted in changes that were manifested by an increased capacity for the uptake of alkali, and a decrease in tensile strength. 4. All four endoglucanases acted synergistically with the exoglucanase [cellobiohydrolase; Wood & McCrae (1972) Biochem. J. 128, 1183-1192] of T. koningii during the early stages of the breakdown of cotton fibre, but only two could produce extensive solubilization of cotton cellulose when acting in admixture with the exoglucanase component. 5. The mode of action of the enzymes is discussed in relation to these synergistic effects. It is suggested that the results are compatible with the interpretation that the 'crystalline' areas of cotton cellulose are hydrolysed only by those endoglucanases capable of forming of forming an enzyme-enzyme complex with the cellobiohydrolase on the surface of the cellulose chains.

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.

The purification and properties of the C 1 component of Trichoderma koningii cellulase.

1. The C(1) component that was isolated from a Trichoderma koningii cellulase preparation (Wood, 1968) by chromatography on DEAE-Sephadex with a salt gradient was still associated with a trace of CM-cellulase activity (determined by reducing-sugar and viscometric methods). 2. Further chromatography on DEAE-Sephadex, with a pH gradient instead of a salt gradient, provided a C(1) component that could still produce reducing sugars from a solution of CM-cellulose (to a very limited extent), but which could no longer decrease the viscosity (i.e. under the assay conditions employed). 3. No evidence for the non-identity of C(1) component and the trace of CM-cellulase activity could be found when electrofocusing was done in a stabilized pH gradient covering three pH units (pH3-6) or, alternatively, only 0.5 pH unit (pH3.72-4.25). 4. The two protein peaks that were separated by electrofocusing in carrier ampholytes covering only 0.5 pH unit (isoelectric pH values of 3.80 and 3.95) were shown to be isoenzymes of the C(1) component: they differed in the extent to which they were associated with carbohydrate (9% and 33%). 5. The purified C(1) component had little ability to attack CM-cellulose or highly ordered forms of cellulose, but degraded phosphoric acid-swollen cellulose readily: cellobiose was the principal product of the hydrolysis (97%). 6. Dewaxed cotton fibre was degraded to the extent of 15% when exposed to high concentrations of C(1) component over a prolonged period: cellobiose was again the principal sugar present in the supernatant (96%). 7. Cellotetraose and cellohexaose were hydrolysed almost exclusively to cellobiose. 8. Evidence indicates that the C(1) component is a beta-1,4-glucan cellobiosylhydrolase.