Ethylene biosynthesis-inducing protein from cellulysin is an endoxylanase

The proteinaceous ethylene biosynthesis-inducing factor (EIF) that was purified from Cellulysin was also shown to contain a xylanase activity. In all nondenaturing protein separation methods employed (Sephacryl S-200 chromatography, and preparative isoelectric focusing and agarose electrophoresis), xylanase activity copurified with the ethylene biosynthesis-inducing activity. Treatment with heat (60°C) or proteases in 8 molar urea inhibited both ethylene-inducing and xylanase activities. Antibodies raised against purified EIF, which contains three polypeptides of 18, 14, and 10 kilodaltons, immunoprecipitated both ethylene biosynthesis-inducing and xylanase activities. The purified EIF contained no detectable cellulase, polygalacturonase, or protease activity. Other hydrolytic activities as estimated by using p-nitrophenyl derivatives of several sugars as substrates also were not detected. Different commercially available hydrolytic enzyme preparations were tested for both ethylene biosynthesis-inducing and xylanase activities. All enzymes tested contained xylanase activity, but only a few induced ethylene biosynthesis. Western blots of proteins separated by SDS-PAGE, using antibodies prepared against the non-denatured purified EIF, revealed two major bands of about 18 and 14 kilodaltons in EIF. These antibodies seem to be specific for these proteins from Trichoderma viride, because there was little cross-reactivity with the other proteins in Cellulysin and other commercial enzyme preparations. Based on these data, we suggest that EIF contains a specific xylanase activity which is involved in inducing ethylene biosynthesis.     

Expression of the xylan-degrading genes of Bacillus pumilus IPO in Saccharomyces cerevisiae

Xylanase (xynA) and β-xylosidase (xynB) genes of Bacillus pumilus were expressed in Saccharomyces cerevisiae by using the GAP (glyceraldehyde-3-phosphate dehydrogenase) promoter of S. cerevisiae. Yeast cells harboring a plasmid pNAX2 containing xynA produced xylanase in the cytoplasm of the cell to an extent as much as 5% of the total soluble protein in the cell extract. Xylanase produced in yeast had an extra methionine at the N-terminus, but had the same specific activity as that produced by B. pumilus IPO. The xylanase in the yeast was not glycosylated and was immunologically identical to that of B. pumilus IPO. Yeast cells harboring a plasmid pYXB containing xynB produced β-xylosidase in the cytoplasm of the cell (3% of the total soluble protein). β-Xylosidase purified from the yeast strain exhibited specific activity nearly equal to the value of enzyme purified from B. pumilus, and had an N-terminal sequence identical to the sequence of the enzyme from B. pumilus.     

Production, Partial Purification and Characterization of Extracelluar Xylanase from Chaetomium globosum

Culture conditions for efficient production of extracellular xylanase by fungus, Chaetomium globosum isolate Cg2, have been standardized. Further, xylanase has been partially purified and characterized. Xylanase activity was maximum after 9 days of incubation when amended in medium with 1.5 % xylan as carbon source and 0.6% NH₄H₂PO₄ as nitrogen source. Partial purification of the xylanase was accomplished by ammonium sulphate precipitation, followed by further purification by anion exchange chromatography on DEAE-Sephadex A-50 column. The partially purified enzyme was electrophoresed on SDS-PAGE and a single band produced corresponded to molecular weight, 32 kD. The optimum temperature and pH for maximum activity of purified xylanase were 30°C and 5.5, respectively. Both the purified xylanase and culture filtrate have shown the antifungal activity against Bipolaris sorokiniana, a causal organism of spot blotch of wheat. Purified xylanase at 100 μg ml^?1 concentration caused 100 per cent inhibition of conidia germination of B. sorokiniana, whereas the culture filtrate was able to inhibit germination up to 67.5 per cent.     

Production and purification of high levels of cellulase-free bacterial xylanase by Bacillus sp. SV-34S using agro-residue

Xylanase produced from the isolated bacterial strain Bacillus sp. SV-34S showed a 8.74-fold increase in enzyme activity under optimized submerged fermentation conditions. Cultivation using wheat bran as the carbon source and beef extract and (NH₄)H₂PO₄ as the nitrogen source resulted in productivity of 3,454.01 IU/mL xylanase. Xylanase was purified by 12.94-fold, with a recovery of 13.4 % and a specific activity of 3417.2 IU/mg protein, employing ammonium sulphate fractionation followed by cation-exchange chromatography using CM-Sephadex C-50 column chromatography, with a product of 27 kDa. The purified xylanase showed an optimum temperature and pH of 50 °C and 6.5, respectively although it was active even at pH 11.0. The thermostability study revealed that Bacillus sp. SV-34S was thermotolerant, being stable up to 50 °C; the residual activity at 55 and 60 °C was 96 and 93 %, respectively. The enzyme was stable between pH 6.0 and 8.0, although it retained >100 % activity at pH 8.0 and 9.0, respectively, following pre-incubation for 24 h. Xylanase activity was inhibited by various metal ions added to the assay mixture, with maximum inhibition observed in the presence of HgCl₂. The K_m and V_max values of the purified xylanase using birch wood xylan as substrate were 3.7 mg/mL and 133.33 IU/mL, respectively. The isolated bacterial strain produced high levels of extremophilic cellulase-free xylanase. The fact that it can be used in crude form and that it can be produced cheaply with renewable carbon sources make the process economically feasible. The characteristics of the purified enzyme suggest its potential application in industries such as the paper and pulp industry.     

Production of a bacterial thermophilic xylanase inSaccharomyces cerevisiae

ThexynA gene (encoding xylanase) from the obligately anaerobic thermophilic bacteriumCaldocellum saccharolyticum has been inserted into the yeast expression vector, pFLAGU2. Yeast cells containing this vector were able to produce and secrete active xylanase into the growth medium. Xylanase was purified by the use of an affinity column specific for a rare peptide sequence fused to the N-terminus of the xylanase. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the purified fractions revealed that the enzyme had been fortuitously glycosylated. The specific activity of the purified xylanase was found to be 90 international units/mg protein. The amount of xylanase secreted into the surrounding medium was approximately 10 mg/l.     

Production, characterization, and partial amino acid sequence of xylanase A from Schizophyllum commune.

Xylanase A, one of several extracellular xylanases produced by Schizophyllum commune strain Delmar when grown in submerged culture with spruce sawdust as carbon source, was purified 43-fold in 25% yield with respect to total xylanase activity. Although some polysaccharide was strongly bound to the purified enzyme, the complex could be dissociated by sodium dodecyl sulfate and appeared homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the protein, calculated from the electrophoretic mobility, was 33,000. The molecular activity of the purified xylanase A, determined with soluble larch xylan as substrate, was 1.4 X 10(5) min-1, with xylobiose and xylose as the major products. The enzyme had a pH optimum of 5.0 and a temperature optimum of 55 degrees C in 10-min assays. The acid hydrolysate of xylanase A was rich in aspartic acid and aromatic amino acids. The sequence of 27 residues at the amino terminus showed no homology with known sequences of other proteins.     

Production, characterization, and partial amino acid sequence of xylanase A from Schizophyllum commune.

Xylanase A, one of several extracellular xylanases produced by Schizophyllum commune strain Delmar when grown in submerged culture with spruce sawdust as carbon source, was purified 43-fold in 25% yield with respect to total xylanase activity. Although some polysaccharide was strongly bound to the purified enzyme, the complex could be dissociated by sodium dodecyl sulfate and appeared homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the protein, calculated from the electrophoretic mobility, was 33,000. The molecular activity of the purified xylanase A, determined with soluble larch xylan as substrate, was 1.4 X 10(5) min-1, with xylobiose and xylose as the major products. The enzyme had a pH optimum of 5.0 and a temperature optimum of 55 degrees C in 10-min assays. The acid hydrolysate of xylanase A was rich in aspartic acid and aromatic amino acids. The sequence of 27 residues at the amino terminus showed no homology with known sequences of other proteins.     

Isolation and Characterization of Xylan-Degrading Strains of Butyrivibrio fibrisolvens from a Napier Grass-Fed Anaerobic Digester

Six new xylanolytic bacterial strains have been isolated from a Napier grass-fed anaerobic digester. These strains were identified as Butyrivibrio fibrisolvens and were similar in many respects to ruminal isolates described previously. The new isolates exhibited a high degree of DNA homology with several ruminal strains of B. fibrisolvens. Xylan or xylose was required to induce the production of enzymes for xylan degradation, xylanase and xylosidase. Production of these enzymes was repressed in the presence of glucose. Xylanase activity was predominantly extracellular, while that of xylosidases was cell associated. The new isolates of B. fibrisolvens grew well in defined medium containing xylan as the sole carbon source and did not produce obvious slime or capsular layers. These strains may be useful for future genetic investigations.     

Identification of GH10 xylanases in strains 2 and Mz5 of Pseudobutyrivibrio xylanivorans

Genes encoding glycosyl hydrolase family 11 (GH11) xylanases and xylanases have been identified from Pseudobutyrivibrio xylanivorans. In contrast, little is known about the diversity and distribution of the GH10 xylanase in strains of P. xylanivorans. Xylanase and associated activities of P. xylanivorans have been characterized in detail in the type strain, Mz5. The aim of the present study was to identify GH10 xylanase genes in strains 2 and Mz5 of P. xylanivorans. In addition, we evaluated degradation and utilization of xylan by P. xylanivorans 2 isolated from rumen of Creole goats. After a 12-h culture, P. xylanivorans 2 was able to utilize up to 53 % of the total pentose content present in birchwood xylan (BWX) and to utilize up to 62 % of a ethanol-acetic acid-soluble fraction prepared from BWX. This is the first report describing the presence of GH10 xylanase-encoding genes in P. xylanivorans. Strain 2 and Mz5 contained xylanases which were related to GH10 xylanase of Butyrivibrio sp. Identifying xylanase-encoding genes and activity of these enzymes are a step toward understanding possible functional role of P. xylanivorans in the rumen ecosystem and contribute to providing an improved choice of enzymes for improving fiber digestion in ruminant animals, agricultural biomass utilization for biofuel production, and other industries.     

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

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.     

Purification and characterization of a thermophilic alkaline xylanase from thermoalkaliphilic Bacillus sp. strain TAR-1

Thermoalkaliphilic Bacillus sp. strain TAR-1 isolated from soil produced an extracellular xylanase. The enzyme (xylanase R) was purified to homogeneity by ammonium sulfate fractionation and anion-exchange chromatography. The molecular mass of xylanase R was 40 kDa and the isoelectric point was 4.1. The enzyme was most active over the range of pH 5.0 to 10.0 at 50°C. The optimum temperatures for activity were 75°C at pH 7.0 and 70°C at pH 9.0. Xylanase R was stable up to 65°C at pH 9.0 for 30 min in the presence of xylan. Mercury(ll) ion at 1 mM concentration abolished all the xylanase activity. The predominant products of xylan-hydrolysate were xylobiose, xylotriose, and higher oligosaccharides, indicating that xylanase R was an endo-acting enzyme. Xylanase R had a K_m of 0.82 mg/ml and a V_max of 280 μmol min⁻¹ mg⁻¹ for xylan at 50°C and pH 9.0.     

High-level expression of a xylanase gene from the thermophilic fungus Paecilomyces thermophila in Pichia pastoris

A xylanase gene from Paecilomyces thermophila was functionally expressed in Pichia pastoris. The recombinant xylanase (xynA) was predominantly extracellular; in a 5?l fermentor culture, the total extracellular protein was 8.1?g?l^?1 with an activity of 52,940?U?ml^?1. The enzyme was purified to homogeneity with a recovery of 48?%. The recombinant xynA was optimally active at 75?°C, as measured over 10?min, and at pH 7. The enzyme was stable up to 80?°C for 30?min. It hydrolyzed birchwood xylan, beechwood xylan and xylooligosaccharides to produce xylobiose and xylotriose as the main products.     

Xylanases of thermophilic bacteria from Icelandic hot springs

Thermophilic, aerobic bacteria isolated from Icelandic hot springs were screened for xylanase activity. Of 97 strains tested, 14 were found to be xylanase positive. Xylanase activities up to 12 nkat/ml were produced by these strains in shake flasks on xylan medium. The xylanases of the two strains producing the highest activities (ITI 36 and ITI 283) were similar with respect to temperature and pH optima (80°C and pH 8.0). Xylanase production of strain ITI 36 was found to be induced by xylan and xylose. Xylanase activity of 24 nkat/ml was obtained with this strain in a laboratory-scale-fermentor cultivation on xylose medium. -Xylosidase activity was also detected in the culture filtrate. The thermal half-life of ITI 36 xylanase was 24 h at 70°C. The highest production of sugars from hydrolysis of beech xylan was obtained at 70°C, although xylan depolymerization was detected even up to 90°C. Correspondence to: M. Rättö     

Thermostable xylanase inhibits and disassembles Pseudomonas aeruginosa biofilms

Pseudomonas aeruginosa biofilms are problematic and play a critical role in the persistence of chronic infections because of their ability to tolerate antimicrobial agents. In this study, various cell-wall degrading enzymes were investigated for their ability to inhibit biofilm formation of two P. aeruginosa strains, PAO1 and PA14. Xylanase markedly inhibited and detached P. aeruginosa biofilms without affecting planktonic growth. Xylanase treatment broke down extracellular polymeric substances and decreased the viscosity of P. aeruginosa strains. However, xylanase treatment did not change the production of pyochelin, pyocyanin, pyoverdine, the Pseudomonas quinolone signal, or rhamnolipid. In addition, the anti-biofilm activity of xylanase was thermally stable for > 100 days at 45°C. Also, xylanase showed anti-biofilm activity against one methicillin-resistance Staphylococcus aureus and two Escherichia coli strains.     

Production of xylanase by an alkaline-tolerant marine-derived Streptomyces viridochromogenes strain and improvement by ribosome engineering

Xylanase is the enzyme complex that is responsible for the degradation of xylan; however, novel xylanase producers remain to be explored in marine environment. In this study, a Streptomyces strain M11 which exhibited xylanase activity was isolated from marine sediment. The 16S rDNA sequence of M11 showed the highest identity (99 %) to that of Streptomyces viridochromogenes. The xylanase produced from M11 exhibited optimum activity at pH 6.0, and the optimum temperature was 70 °C. M11 xylanase activity was stable in the pH range of 6.0–9.0 and at 60 °C for 60 min. Xylanase activity was observed to be stable in the presence of up to 5 M NaCl. Antibiotic-resistant mutants of M11 were isolated, and among the various antibiotics tested, streptomycin showed the best effect on obtaining xylanase overproducer. Mutant M11-1(10) isolated from 10 μg/ml streptomycin-containing plate showed 14 % higher xylanase activities than that of the wild-type strain. An analysis of gene rpsL (encoding ribosomal protein S12) showed that rpsL from M11-1(10) contains a K88R mutation. This is the first report to show that marine-derived S. viridochromogenes strain can be used as a xylanase producer, and utilization of ribosome engineering for the improvement of xylanase production in Streptomyces was also first successfully demonstrated.     

Purification and characterisation of a xylanase from Thermomyces lanuginosus and its functional expression by Pichia pastoris

A xylanase produced by Thermomyces lanuginosus 195 by solid state fermentation (SSF) was purified 9.3-fold from a crude koji extract, with a 7.6% final yield. The purified xylanase (with an estimated mass of 22 kDa by SDS-PAGE) retained 18% relative activity when treated for 10 min at 100 °C and approximately 90% relative activity when incubated at pH values ranging from 6 to 10. Xylanase activity in the purified preparation was significantly enhanced following treatment with manganese and potassium chlorides (p < 0.05) but significantly reduced by calcium, cobalt and iron (p < 0.05). The purified enzyme was also shown to be exclusively xylanolytic. The gene encoding xylanase activity from T. lanuginosus 195 was functionally expressed by Pichia pastoris. MALDI-ToF mass spectrometry and zymography were employed to confirm functional recombinant expression. Maximum xylanase titres were achieved following 120 h induction of the recombinant culture, yielding 26.8 U/mL. Achieving functional protein expression facilitates future efforts to optimise the cultivation conditions for heterologous xylanase production.     

Purification and characterization of a moderately thermostable xylanase from Bacillus sp. strain SPS-0

A Bacillus spp. strain SPS-0, isolated from a hot spring in Portugal, produced an extracellular xylanase upon growth on wheat bran arabinoxylan. The enzyme was purified to homogeneity by ammonium sulfate precipitation, anion exchange, gel filtration, and affinity chromatography. The optimum temperature and pH for activity was 75 degrees C and 6.0. Xylanase was stable up to 70 degrees C for 4 h at pH 6.0 in the presence of xylane. Xylanase was completely inhibited by the Hg(2+) ions. beta-Mercaptoethanol, dithiothreitol, and Mn(2+) stimulated the xylanase activity. The products of birchwood xylan hydrolysis were xylose, xylobiose, xylotriose, and xylotetraose. Kinetic experiments at 60 degrees C and pH 6.0 gave V(max) and K(m)values of 2420 nkat/mg and 0.7 mg/ml.     

Characterization of a xylanase from the newly isolated thermophilic Thermomyces lanuginosus CAU44 and its application in bread making

AIMS: A xylanase from the newly isolated thermophilic fungus, Thermomyces lanuginosus CAU44, was characterized and evaluated for its suitability in bread making. METHODS AND RESULTS: Xylanase was purified 3.5-fold to homogeneity with a recovery yield of 32.8%. It appeared as a single protein band on SDS-PAGE gel with a molecular mass of c. 25.6 kDa. The purified xylanase had an optimum pH of 6.2, and it was stable over pH 5.6-10.3. The optimal temperature of xylanase was 75 degrees C and it was stable up to 65 degrees C at pH 6.2. Study was further carried out to investigate the effect of the purified xylanase on the properties of wheat bread and its staling during storage. CONCLUSIONS: The purified xylanase from T. lanuginosus CAU44 was stable up to 65 degrees C and had a broad pH range. The presence of thermostable xylanase during bread making led to an improvement of the specific bread volume and better crumb texture. Besides, addition of xylanase provided an anti-staling effect. SIGNIFICANCE AND IMPACT OF THE STUDY: The xylanase from the newly isolated Thermomyces lanuginosus CAU44 shows great promise as a processing aid in the bread-making industry.     

Production of alkaliphilic, halotolerent, thermostable cellulase free xylanase by Bacillus halodurans PPKS-2 using agro waste: single step purification and characterization

The alkaliphilic Bacillus halodurans strain PPKS-2 was shown to produce extracellular alkaliphilic, thermostable and halotolerent xylanase. The culture conditions for xylanase production were optimized with respect to pH, temperature, NaCl and inexpensive agro waste as substrates. Xylanase yield was enhanced more than four fold in the presence of 1% corn husk and 0.5% peptone or feather hydrolysate at pH 11 and 37°C. Xylanase was purified to 11.8-fold with 8.7% yield by using traditional chromatographic methods whereas the same enzyme purified to 20-fold with 72% yield by using corn husk as ligand. Its molecular mass was estimated to be 24 kDa by SDS–PAGE. The xylanase had maximal activity at pH 11 and 70°C. The enzyme was active over broad range, 0–20% sodium chloride. The enzyme was thermostable retaining 100% of the original activity at 70°C for 3 h. The apparent K _m values for oat spelt xylan and brichwood xylan were 4.1 and 4.4 mg/ml respectively. The deduced internal amino acid sequence of PPKS-2 xylanase resembled the sequence of β-1,4-endoxylanase, which is member of glycoside hydrolase family 11.     

The rb Mutation of Peas Causes Structural and Regulatory Changes in ADP Glucose Pyrophosphorylase from Developing Embryos

A mutation at the rb locus of pea (Pisum sativum L.) alters the shape, reduces the starch content, and increases the lipid and sucrose contents of the seed. These effects are probably all consequences of a reduction of up to 40-fold in the maximum catalytic activity of ADP glucose pyrophosphorylase in the developing embryo of the mutant relative to the wild type. We have investigated how the mutation brings about this reduction in activity. The purified enzyme from mutant embryos has a specific activity about 10-fold lower than that from wild-type embryos, and it is much more sensitive to the effectors inorganic phosphate and 3-phosphoglycerate than the wild-type enzyme. Both wild-type and mutant enzymes consist of polypeptides of around 50 kilodaltons. One of the polypeptides of the purified wild-type enzyme is missing from the mutant enzyme. We deduce that in the wild-type embryo this protein may interact with other subunits to confer a high specific activity and a low susceptibility to effectors on the enzyme.