Mode of action and properties of xylanase and beta-Xylosidase from Neurospora crassa

Extracellular beta-xylosidase (1,4-beta-D-xylan xylohydrolase, EC 3.2.1.37) from culture filtrates of Neurospora crassa was purified to homogeneity by preparative isoelectric focusing followed by gel electrophoresis. The molecular weight of the purified xylosidase was 83,000 D and the K(m) on p-nitrophenyl-beta-D-xyloside was 0.047mM. The homogeneous xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) and beta-xylosidase showed differences in their mode of action towards xylooligosaccharides. The degree of hydrolysis of D-xylan by xylanase of N. crassa was 18%. Supplementation of beta-xylosidase from the same organism resulted in 48% hydrolysis. The synergistic effect was more pronounced, with the hydrolysis of 68%, when a homogeneous preparation of beta-xylosidase from Sclerotium rolfsii was added to the saccharification system.     

Purification and characterization of endo-xylanases from Aspergillus niger. III. An enzyme of pl 3.65

An endo-xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) from Aspergillus niger was purified to homogeneity by chromatography with Ultrogel AcA 54, SP-Sephadex C-25 at pH 4.5, DEAE-Sephadex A-25 at pH 5.4, Sephadex G-50, and DEAE-Sephadex A-25 at pH 5.15. The enzyme was active on soluble xylan, on insoluble xylan only after arabinosyl-initiated branch points were removed, and on xylooligosaccharides longer than xylotetraose. There was slight activity on carboxymethyl-cellulose, arabinogalactan, glucomannan, and p-nitrophenyl-beta-D-glucopyranoside. The main products of the hydrolysis of soluble and insoluble xylan were oligosaccharides of intermediate length, especially the tri- and pentasaccharides. The isoelectric point of the enzyme was 3.65. It had a molecular weight of 2.8 x 10(4) by SDS-gel electrophoresis, and was high in acidic amino acids but low in those containing sulfur. Highest activity in a 20-min assay at pH 5 was between 40 and 45 degrees C, with an activation energy up to 40 degrees C of 11.1 kJ/mol. The optimum pH for activity was at 5.0. The enzyme was strongly activated by Ca(2+).     

Purification, characterization and partial amino acid sequences of a xylanase produced by Penicillium chrysogenum.

An extracellular xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8, endo 1,4-beta-xylanase) was found to be the major protein in the culture filtrate of Penicillium chrysogenum when grown on 1% xylan. In contrast to other microorganism no xylanase multiplicity was found in P. chrysogenum under the conditions used. This enzyme was purified to homogeneity by high performance anion-exchange and size-exclusion chromatography. It had an M(r) of 35,000 as estimated by SDS-PAGE and was shown to be active as a monomer. No glycosylation of the protein could be detected neither by a sensitive glycostain nor by enzymatic deglycosylation studies. The enzyme hydrolyzed oat spelt and birchwood xylan randomly, yielding xylose and xylobiose as major end products. It had no cellulase, CMCase, beta-xylosidase or arabinogalactanase activity but acted on p-nitrophenylcellobioside. The pH and temperature optima for its activity were pH 6.0 and 40 degrees C, respectively. Eight peptides obtained after endoproteinase LysC digestion of xylanase have been sequenced, six of them showed considerable amino acid similarity to glucanases and high M(r)/acidic xylanases from different bacteria, yeasts and fungi.     

Production and Properties of Extracellular Endoxylanase from Neurospora crassa

Neurospora crassa 870 produced 14 and 0.025 U of extracellular xylanase (1,4-beta-d-xylan xylanohydrolase; EC 3.2.1.8) and beta-xylosidase (1,4-beta-xylan xylohydrolase; EC 3.2.1.37) per ml, respectively, in 4 days when commercial xylan was used as a carbon source. The effects of pH and carbon sources on xylanase production by N. crassa are discussed. Two xylanases (I and II) were purified and had pI values of 4.8 and 4.5 and molecular weights of 33,000 and 30,000. The maximum degree of hydrolysis of xylan by the extracellular culture broth was 66% in 4 h. The end products of xylan hydrolysis by xylanase I and II showed the presence of xylose, xylobiose, xylotriose, xylotetraose, xylopentose, and arabinose, indicating that they are endoxylanases capable of hydrolyzing 1,3-alpha-l-arabinofuranosyl branch points. Both xylanases showed activity toward carboxymethyl cellulose but no activity toward para-nitrophenyl-beta-d-xyloside or laminarin. Xylanase I showed appreciable activity toward para-nitrophenyl-beta-d-glucoside, whereas xylanase II was inactive.     

Production of xylanase by the ruminal anaerobic fungus Neocallimastix frontalis.

Xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) production was investigated in the ruminal anaerobic fungus Neocallimastix frontalis. The enzyme was released principally into the culture fluid and had pH and temperature optima of 5.5 and 55 degrees C, respectively. In the presence of low concentrations of substrate, the enzyme was stabilized at 50 degrees C. Xylobiose was the principal product of xylanase action, with lesser amounts of longer-chained xylooligosaccharides. No xylose was detected, indicating that xylobiase activity was absent. Activities of xylanase up to 27 U ml-1 (1 U represents 1 micromol of xylose equivalents released min-1) were obtained for cultures grown on xylan (from oat spelt) at 2.5 mg ml-1 in shaken cultures. No growth occurred in unshaken cultures. Xylanase production declined with elevated concentrations of xylan (less than 2.5 mg ml-1), and this was accompanied by an accumulation of xylose and, to a lesser extent, arabinose. Addition of either pentose to cultures grown on low levels of xylan in which neither sugar accumulated suppressed xylanase production, and in growth studies with the paired substrates xylan-xylose, active production of the enzyme occurred during growth on xylan only after xylose had been preferentially utilized. When cellobiose, glucose, and xylose were tested as growth substrates for the production of xylanase (each initially at 2.5 mg ml-1), they were found to be less effective than xylan, and use of xylan from different origins (birch wood or larch wood) as the growth substrate or in the assay system resulted in only marginal differences in enzyme activity. However, elevated production of xylanase occurred during growth on crude hemicellulose (barley straw leaf). The results are discussed in relation to the role of the anaerobic fungi in the ruminal ecosystem, and the possible application of the enzyme in bioconversion processes is also considered.     

Low-molecular-weight xylanase from Trichoderma viride.

An endo-1,4-beta-xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) has been isolated from a commercial preparation of Trichoderma viride. The molecular weight was 22,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the pI value was 9.3. The xylanase was a true xylanase without cellulase activity. When the N-terminal amino acid sequence of the first 50 residues was compared with that of a xylanase from Schizophyllum commune, strong evidence for homology was found, with more than 50% amino acid identity. T. viride xylanase also possessed extensive identity with a proposed amino-terminal consensus sequence of xylanases from bacteria.     

Production of xylanase by the ruminal anaerobic fungus Neocallimastix frontalis

Xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) production was investigated in the ruminal anaerobic fungus Neocallimastix frontalis. The enzyme was released principally into the culture fluid and had pH and temperature optima of 5.5 and 55 degrees C, respectively. In the presence of low concentrations of substrate, the enzyme was stabilized at 50 degrees C. Xylobiose was the principal product of xylanase action, with lesser amounts of longer-chained xylooligosaccharides. No xylose was detected, indicating that xylobiase activity was absent. Activities of xylanase up to 27 U ml-1 (1 U represents 1 micromol of xylose equivalents released min-1) were obtained for cultures grown on xylan (from oat spelt) at 2.5 mg ml-1 in shaken cultures. No growth occurred in unshaken cultures. Xylanase production declined with elevated concentrations of xylan (less than 2.5 mg ml-1), and this was accompanied by an accumulation of xylose and, to a lesser extent, arabinose. Addition of either pentose to cultures grown on low levels of xylan in which neither sugar accumulated suppressed xylanase production, and in growth studies with the paired substrates xylan-xylose, active production of the enzyme occurred during growth on xylan only after xylose had been preferentially utilized. When cellobiose, glucose, and xylose were tested as growth substrates for the production of xylanase (each initially at 2.5 mg ml-1), they were found to be less effective than xylan, and use of xylan from different origins (birch wood or larch wood) as the growth substrate or in the assay system resulted in only marginal differences in enzyme activity. However, elevated production of xylanase occurred during growth on crude hemicellulose (barley straw leaf). The results are discussed in relation to the role of the anaerobic fungi in the ruminal ecosystem, and the possible application of the enzyme in bioconversion processes is also considered.     

Low-molecular-weight xylanase from Trichoderma viride

An endo-1,4-beta-xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) has been isolated from a commercial preparation of Trichoderma viride. The molecular weight was 22,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the pI value was 9.3. The xylanase was a true xylanase without cellulase activity. When the N-terminal amino acid sequence of the first 50 residues was compared with that of a xylanase from Schizophyllum commune, strong evidence for homology was found, with more than 50% amino acid identity. T. viride xylanase also possessed extensive identity with a proposed amino-terminal consensus sequence of xylanases from bacteria.     

Purification, characterization, and mode of action of endoxylanases 1 and 2 from Fibrobacter succinogenes S85.

Two different endoxylanases (1,4-beta-D-xylan xylanohydrolases, EC 3.2.1.8), designated 1 and 2, have been purified by column chromatography to apparent homogeneity from the nonsedimentable extracellular culture fluid of the strictly anaerobic, ruminal bacterium Fibrobacter succinogenes S85 grown on crystalline cellulose. Endoxylanases 1 and 2 were shown to be basic proteins of 53.7 and 66.0 kDa, respectively, with different pH and temperature optima, as well as different substrate hydrolysis characteristics. The Km and Vmax values with water-soluble oat spelts xylan as substrate were 2.6 mg ml-1 and 33.6 mumol min-1 mg-1 for endoxylanase 1 and 1.3 mg ml-1 and 118 mumol min-1 mg-1 for endoxylanase 2. Endoxylanase 1, but not endoxylanase 2, released arabinose from water-soluble oat spelts xylan and rye flour arabinoxylan, but not from arabinan, arabinogalactan, or aryl-alpha-L-arabinofuranosides. With an extended hydrolysis time, endoxylanase 1 released 62.5 and 50% of the available arabinose from water-soluble oat spelts xylan and rye flour arabinoxylan, respectively. Endoxylanase 1 released arabinose directly from the xylan backbone, and this preceded hydrolysis of the xylan to xylooligosaccharides. Endoxylanase 2 showed significant activity against carboxymethyl cellulose but was unable to substantially hydrolyze acid-swollen cellulose. Both enzymes were endo-acting, as revealed by their hydrolysis product profiles on water-soluble xylan and xylooligosaccharides. Because of their unique hydrolytic properties, endoxylanases 1 and 2 appear to have strategic roles in plant cell wall digestion by F. succinogenes in vivo.     

Purification, characterization, and mode of action of endoxylanases 1 and 2 from Fibrobacter succinogenes S85.

Two different endoxylanases (1,4-beta-D-xylan xylanohydrolases, EC 3.2.1.8), designated 1 and 2, have been purified by column chromatography to apparent homogeneity from the nonsedimentable extracellular culture fluid of the strictly anaerobic, ruminal bacterium Fibrobacter succinogenes S85 grown on crystalline cellulose. Endoxylanases 1 and 2 were shown to be basic proteins of 53.7 and 66.0 kDa, respectively, with different pH and temperature optima, as well as different substrate hydrolysis characteristics. The Km and Vmax values with water-soluble oat spelts xylan as substrate were 2.6 mg ml-1 and 33.6 mumol min-1 mg-1 for endoxylanase 1 and 1.3 mg ml-1 and 118 mumol min-1 mg-1 for endoxylanase 2. Endoxylanase 1, but not endoxylanase 2, released arabinose from water-soluble oat spelts xylan and rye flour arabinoxylan, but not from arabinan, arabinogalactan, or aryl-alpha-L-arabinofuranosides. With an extended hydrolysis time, endoxylanase 1 released 62.5 and 50% of the available arabinose from water-soluble oat spelts xylan and rye flour arabinoxylan, respectively. Endoxylanase 1 released arabinose directly from the xylan backbone, and this preceded hydrolysis of the xylan to xylooligosaccharides. Endoxylanase 2 showed significant activity against carboxymethyl cellulose but was unable to substantially hydrolyze acid-swollen cellulose. Both enzymes were endo-acting, as revealed by their hydrolysis product profiles on water-soluble xylan and xylooligosaccharides. Because of their unique hydrolytic properties, endoxylanases 1 and 2 appear to have strategic roles in plant cell wall digestion by F. succinogenes in vivo.     

Purification and characterization of endo-xylanases from Aspergillus niger. I. Two isozymes active on xylan backbones near branch points

Two endo-xylanases (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) were purified to homogeneity from a crude Aspergillus niger pentosanase preparation by Ultrogel AcA 54 gel permeation chromatography, SP-Sephadex C-25 cation exchange chromatography at pH 4.5, Sephadex G-50 gel permeation chromatography, and a second SP-Sephadex C-25 step, this one at pH 5.8. The two xylanases hydrolyzed soluble xylan more rapidly than insoluble branched xylan, but attacked each substance to an equal extent. Their low activity on a linear xylooligosaccharide mixture and absence of activity on insoluble xylan freed of branches suggest that the xylanases require a branch point nearby for significant attack. No xylose or L-arabinose was produced, the major products of low molecular weight being tri- and pentasaccharides and smaller amounts of di-, tetra-, and hexasaccharides. There was low activity on untreated and crystalline cellulose and on carboxymethylcellulose and no activity on other polysaccharides tested. These two xylanases had molecular weights of ca. 1.3 x 10(4) and similar amino acid profiles, high in acidic and low in sulfur-containing residues. Isoelectric points were 8.6 for I and 9.0 for II. Optimum pH values for activity were 6.0 and 5.5, respectively. In a 20-min assay at pH 5.5, each was most active at 45 degrees C, with activation energies up to 40 degrees C of 30.4 and 38.8 kJ/ mol, respectively. Optimum pH levels for stability were 5.0 and 6.0, with half-lives at 60 degrees C and those pHs of 20 and 75 min, respectively.     

Purification, properties, and partial amino acid sequences of thermostable xylanases from Streptomyces thermoviolaceus OPC-520.

Two types of xylanases (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) were isolated from the culture filtrate of a thermophilic actinomycete, Streptomyces thermoviolaceus OPC-520. The enzymes (STX-I and STX-II) were purified by chromatography with DEAE-Toyopearl 650 M, CM-Toyopearl 650 M, Sephadex G-75, Phenyl-Toyopearl 650 M, and Mono Q HR. The purified enzymes showed single bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weights of STX-I and STX-II were 54,000 and 33,000, respectively. The pIs were 4.2 (STX-I) and 8.0 (STX-II). The optimum pH levels for the activity of STX-I and STX-II were pH 7.0. The optimum temperature for the activity of STX-I was 70 degrees C, and that for the activity of STX-II was 60 degrees C. The enzymes were completely inhibited by N-bromosuccinimide. The enzymes degraded xylan, producing xylose and xylobiose as the predominant products, indicating that they were endoxylanases. STX-I showed high sequence homology with the exoglucanase from Cellulomonas fimi (47% homology), and STX-II showed high sequence homology with the xylanase from Bacillus pumilus (46% homology).     

Purification, properties, and partial amino acid sequences of thermostable xylanases from Streptomyces thermoviolaceus OPC-520.

Two types of xylanases (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) were isolated from the culture filtrate of a thermophilic actinomycete, Streptomyces thermoviolaceus OPC-520. The enzymes (STX-I and STX-II) were purified by chromatography with DEAE-Toyopearl 650 M, CM-Toyopearl 650 M, Sephadex G-75, Phenyl-Toyopearl 650 M, and Mono Q HR. The purified enzymes showed single bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weights of STX-I and STX-II were 54,000 and 33,000, respectively. The pIs were 4.2 (STX-I) and 8.0 (STX-II). The optimum pH levels for the activity of STX-I and STX-II were pH 7.0. The optimum temperature for the activity of STX-I was 70 degrees C, and that for the activity of STX-II was 60 degrees C. The enzymes were completely inhibited by N-bromosuccinimide. The enzymes degraded xylan, producing xylose and xylobiose as the predominant products, indicating that they were endoxylanases. STX-I showed high sequence homology with the exoglucanase from Cellulomonas fimi (47% homology), and STX-II showed high sequence homology with the xylanase from Bacillus pumilus (46% homology).     

Direct comparison of the crystal and solution structure of xylanase from Trichoderma longibrachiatum

The small angle X-ray scattering (SAXS) data of xylanase XYNII (endo-1,4-beta-xylan xylanohydrolase EC 3.2.1.8) from Trichoderma longibrachiatum, an enzyme catalysing the reaction of accidental hydrolysis of beta-1,4-D-xylosidic linkages of xylan, were recorded for protein solution using synchrotron radiation. The experimental data were compared with those of theoretical scattering calculated on the basis of the known crystal structure. The radius of gyration measured by SAXS (RG = 1.7 nm) was about 3.5% larger and the maximum dimension in the distance distribution function about 5 % larger than the corresponding values calculated on the basis of the crystal structure.     

Purification and properties of a xylanase from Streptomyces lividans.

An extracellular xylanase produced by a cellulase-negative mutant strain of Streptomyces lividans 1326 was purified to homogeneity. The purified enzyme has an apparent Mr of 43,000 and pI of 5.2. The pH and temperature optima for the activity were 6.0 and 60 degrees C respectively, and the Km and Vmax. values, determined with a soluble oat spelts xylan, were 0.78 mg/ml and 0.85 mmol/min per mg of enzyme. The xylanase showed no activity towards CM-cellulose and p-nitrophenyl beta-D-xyloside. The enzyme degraded xylan, producing mainly xylobiose, a mixture of xylo-oligosaccharides and a small amount of xylose as end products. Its pattern of action on beta-1,4-D-xylan indicates that it is a beta-1,4-endoxylanase (EC 3.2.1.8).     

Purification and characterization of endo-xylanases from Aspergillus niger. II. An enzyme of pl 4.5

A homogeneous endo-xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) was obtained from a crude Aspergillus niger pentosanase by chromatography with Ultrogel AcA 54, SP-Sephadex C-25 at pH 4.5, DEAE-Sephadex A-25 at pH 5.4, Sephadex G-50, and SP-Sephadex C-25 with a gradient from pH 2.8 to pH 4.6. It was much more active on soluble than on insoluble xylan, yielding large amounts of unreacted xylan and a mixture of oligosaccharides with chain lengths from two to six. No xylose or L-arabinose was produced. There was high activity on a xylopentaose through xylononaose mixture, but not on xylobiose, xylotriose, or xylotetraose. The enzyme had slight activity on untreated cellulose, carboxymethylcellulose, and pectin. Molecular weight was ca. 1.4 x 10(4), with an isoelectric point of 4.5 and an amino acid profile high in acidic but low in sulfur-containing residues. In a 25-min assay at pH 4.7, this endo-xylanase was most active at 45 degrees C, with an activation energy from 5 to 35 degrees C of 33.3 kJ/mol. The optimum pH for activity was 4.9. Decay in buffer was first order, with an activation energy at pH 4.7 from 48 to 53 degrees C of 460 kJ/mol. Optimum pH for stability was about 5.6, where the half-life at 48 degrees C in buffer was ca. 40 h.     

Cell wall-associated 1,4-beta-D-xylanase in Cryptococcus albidus var. aerius: in situ characterization of the activity.

1,4-beta-D-Xylanase (1,4-beta-D-xylan xylanohydrolase; EC 3.2.1.8) has been detected in both cell-free extracts and culture fluids of the yeast Cryptococcus albidus var. aerius grown on glucose as the only carbon source. Mild acid treatment of whole cells proved that the enzyme was extracellularly located. The activity remained almost completely linked to the wall after cell breakage, only being liberated in the presence of salt at high concentration. After release, the enzyme became very unstable and so has been characterized in situ in 'permeabilized' cells. The maximum production took place at the beginning of the exponential growth phase. The optimum pH and temperature for activity were 5.0 and 40 degrees C, respectively. The enzyme degraded xylan and xylo-oligosides by an endo-splitting mechanism giving xylobiose, xylotriose and xylose as the main end-products. Activation energy and kinetic constants for xylan degradation were determined. Several metal ions such as Ag+ and Hg2+ inhibited the enzyme. The possible function of this endo-xylanase in Cr. albidus var. aerius is discussed.     

Purification and characterization of xylanases from<Emphasis Type="Italic">Aspergillus giganteus</Emphasis>

A strain of Aspergillus giganteus cultivated in a medium with xylan produced two xylanases (xylanase I and II) which were purified to homogeneity. Their molar mass, estimated by SDS-PAGE, were 21 and 24 kDa, respectively. Both enzymes are glycoproteins with 50 degrees C temperature optimum; optimum pH was 6.0-6.5 for xylanase I and 6.0 for xylanase II. At 50 degrees C xylanase I exhibited higher thermostability than xylanase II. Hg2+, Cu2+ and SDS were strong inhibitors, 1,4-dithiothreitol stimulated the reaction of both enzymes. Both xylanases are xylan-specific; kinetic parameters indicated higher efficiency in the hydrolysis of oat spelts xylan. In hydrolysis of this substrate, xylotriose, xylotetraose and larger xylooligosaccharides were released and hence the enzymes were classified as endoxylanases.     

Xylanase and acetyl xylan esterase activities of XynA,a key subunit of the Clostridium cellulovorans cellulosome for xylan degradation

The Clostridium cellulovorans xynA gene encodes the cellulosomal endo-1,4-beta-xylanase XynA, which consists of a family 11 glycoside hydrolase catalytic domain (CD), a dockerin domain, and a NodB domain. The recombinant acetyl xylan esterase (rNodB) encoded by the NodB domain exhibited broad substrate specificity and released acetate not only from acetylated xylan but also from other acetylated substrates. rNodB acted synergistically with the xylanase CD of XynA for hydrolysis of acetylated xylan. Immunological analyses revealed that XynA corresponds to a major xylanase in the cellulosomal fraction. These results indicate that XynA is a key enzymatic subunit for xylan degradation in C. cellulovorans.     

A novel strain of Streptomyces malaysiensis isolated from Brazilian soil produces high endo-β-1,4-xylanase titres

One hundred and sixty two actinomycete strains isolated from Brazilian soils were screened for xylanase activity, according to the size of the hydrolysis zones observed in oat spelts xylan agar plates. The strain AMT-3, later identified as Streptomyces malaysiensis, was selected as the best producer. In subsequent shake flasks fermentations using growth media contanning 1% (w/v) of either birchwood, or oat spelts xylan, plus organic nitrogen and salts, high endo--1,4-xylanase titres (EC 3.2.1.8) (116 U ml^–1) were observed in the larchwood medium within 6 days. This is the first report concerning xylanase production by streptomyces malaysiensis, which has been recently described as a new species.