Purification and characterization of endo-1,4-β-xylanase from Paecilomyces varioti Bainier

An endo-xylanase (1,4-β-d-xylanxylanohydrolase EC 3.2.1.8) was isolated from the culture filtrate of Paecilomyces varioti Bainier. The enzyme was purified 3.2 fold with a 60% yield by gel filtration and ion exchange chromatography. The purified enzyme had a molecular weight of 25,000 with a sedimentation coefficient of 2.2 S. The isoelectric point of the enzyme was 3.9. The enzyme was obtained in crystalline form. The optimum pH range was 5.5–7.0 and the temperature, 65°C. The Michaelis constant was 2.5 mg larchwood xylan/ml. The enzyme was found to degrade xylan by an endo mechanism producing arabinose, xylobiose, xylo- and arabinosylxylo-oligosaccharides, during the initial stages of hydrolysis. On prolonged incubation, xylotriose, arabinosylxylotriose and xylobiose were the major products with traces of xylotetraose, xylose and arabinose.     

Purification and Properties of beta-1, 4-Xylanase from Aeromonas caviae W-61

Aeromonas caviae W-61, which was isolated from water samples at the Faculty of Agriculture, Tohoku University, produced beta-1, 4-xylanase (1,4-beta-d-xylan xylanohydrolase; EC 3.2.1.8) extracellularly. The xylanase was purified to homogeneity by using DEAE-Sephadex A-50, CM-Sephadex C-50, and Sephadex G-100 column chromatographies. The molecular weight of the purified enzyme was estimated to be 22,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point of the enzyme was 9.2. The optimal pH and temperature for the activity of the enzyme were 7.0 and 55 degrees C, respectively. The enzyme was stable at pH 7.0 at temperatures of up to 50 degrees C. As enzymatic products, various xylo-oligosaccharides such as xylobiose, xylotriose, xylotetraose, and xylopentaose were formed, and only a small amount of xylose was detected. The purified enzyme did not hydrolyze starch, cellulose, carboxymethylcellulose, or beta-1, 3-xylan.     

Xylanase IV, an Exoxylanase of Aeromonas caviae ME-1 Which Produces Xylotetraose as the Only Low-Molecular-Weight Oligosaccharide from Xylan

A novel xylanase (xylanase IV) which produces xylotetraose as the only low-molecular-weight oligosaccharide from oat spelt xylan was isolated from the culture medium of Aeromonas caviae ME-1. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the xylanase IV molecular weight was 41,000. Xylanase IV catalyzed the hydrolysis of oat spelt xylan, producing exclusively xylotetraose. The acid hydrolysate of the product gave d-xylose. The enzyme did not hydrolyze either p-nitrophenyl-(beta)-d-xyloside, small oligosaccharides (xylobiose and xylotetraose), or polysaccharides, such as starch, cellulose, carboxymethyl cellulose, laminarin, and (beta)-1,3-xylan.     

Purification and characterization of a xylobiose- and xylose-producing endo-xylanase from Aspergillus niger

A xylanase from a commercial Aspergillus niger pentoglycanase was purified to homogeneity by column chromatography on Ultrogel AcA 54, SP-Sephadex, Sephadex G-50, and SP-Sephadex. The enzyme hydrolyzed xylotriose slowly to xylose and xylobiose, and xylotetraose and higher xylo-oligosaccharides rapidly to mixtures of smaller xylo-oligosaccharides, with xylobiose and xylose being the preponderant final products. The anomeric configuration of the products was inverted, in contrast to the behavior of most other carbohydrases that initially produce mixtures of oligosaccharides. This enzyme is a glycoprotein having an amino acid composition high in acidic residues. Its molecular weight is 20,800 and its isoelectric point is at pH 6.7. Optimal pH values for activity and stability are between 4 and 6 and, in a 20-min assay, maximal activity is attained at 55°.     

Purification and Characterisation of Xylanolytic Enzymes of a Cellulase-free Thermophilic strain of Clostridium absonum CFR-702

Two endo-β-1-4-xylanases (EC 3.2.1.8), xylanase-I and xylanase-II, were purified fromClostridium absonum CFR-702 by ammonium sulphate precipitation and chromatographed on DEAE-Cellulose and phenyl-Sepharose. The enzymes in sodium dodecyl sulphate polyacrylamide gels resolved as proteins corresponding to molecular mass 150 and 95 kDa for xylanase-I and xylanase-II, respectively. The optimum pH and temperature ranges for the enzyme activities on birchwood xylan were between 6.5 and 7.5 and 75°C for xyl-I and 7.5 and 80°C for xyl-II. Xyl-I was stable up to 60°C whereas xyl-II was stable at 50°C. Both the enzymes liberated xylobiose, xylotriose and xylotetraose from birchwood xylan. Xyl-I and xyl-II with birchwood xylan had K_mvalues of 1.1 and 1.4%, and V_maxvalues of 454.54 and 363.63 μmol/min/mg protein respectively.     

Total Hydrolysis of Xylotetraose and Xylobiose by Soluble and Cross-linked Crystalline Xylanase II from Trichoderma reesei

The ability of Trichoderma reesei xylanase II (EC 3.2.1.8) to hydrolyse the small xylo-oligomer substrates, xylotetraose and xylobiose, was studied. Xylanase was used in both soluble and cross-linked enzyme crystal (CLEC) form. Hydrolysis reactions with crystalline xylanase cross-linked with glutaraldehyde and lysine were performed in a column reactor. By using appropriate combination of column packing length and flow rate, xylotetraose and xylobiose (initial concentrations 10 mg ml &#109 1 ) were hydrolysed completely to xylose in less than 1 h. The observed reaction rate in the column depended substantially on the flow rate of the eluent, probably due to an enhanced mass-transfer with higher flow rates. With soluble xylanase, using extended reaction times of 24 h and extremely high enzyme/substrate ratios of 20 (w/w) or above, the hydrolysis reaction reached completion with both xylotetraose and xylobiose as substrates. Even with the lowest flow rate, the reaction in the column appeared to be faster than soluble enzyme hydrolysis with comparable enzyme/substrate ratios.     

Total Hydrolysis of Xylotetraose and Xylobiose by Soluble and Cross-linked Crystalline Xylanase II from Trichoderma reesei

The ability of Trichoderma reesei xylanase II (EC 3.2.1.8) to hydrolyse the small xylo-oligomer substrates, xylotetraose and xylobiose, was studied. Xylanase was used in both soluble and cross-linked enzyme crystal (CLEC) form. Hydrolysis reactions with crystalline xylanase cross-linked with glutaraldehyde and lysine were performed in a column reactor. By using appropriate combination of column packing length and flow rate, xylotetraose and xylobiose (initial concentrations 10 mg ml -1 ) were hydrolysed completely to xylose in less than 1 h. The observed reaction rate in the column depended substantially on the flow rate of the eluent, probably due to an enhanced mass-transfer with higher flow rates. With soluble xylanase, using extended reaction times of 24 h and extremely high enzyme/substrate ratios of 20 (w/w) or above, the hydrolysis reaction reached completion with both xylotetraose and xylobiose as substrates. Even with the lowest flow rate, the reaction in the column appeared to be faster than soluble enzyme hydrolysis with comparable enzyme/substrate ratios.     

XynX, a possible exo-xylanase of Aeromonas caviae ME-1 that produces exclusively xylobiose and xylotetraose from xylan.

A gene, xynX, encoding a novel xylanase, was cloned from Aeromonas caviae ME-1. This gene encoded an enzyme that was constituted of 334 amino acid residues (38,580 Da) and was similar in sequence to Family 10 (Family F) beta-1,4 endo-xylanases. XynX produced only xylobiose and xylotetraose from birch wood xylan, and xylotriose, xylopentaose, and higher oligosaccharides were not detected in the TLC analysis. We designated it as X2/X4-forming xylanase. This enzyme does not have transglycosylation activity. These data suggested that this enzyme is a possible exo-xylanase. According to homology modeling, the enzyme has a ring-shaped (alpha/beta)8 barrel (TIM barrel) structure, typical of Family 10 endo-xylanases, with the extraordinary feature of a longer bottom-loop structure.     

Isolation and analysis of a gene encoding α-glucuronidase, an enzyme with a novel primary structure involved in the breakdown of xylan

This is the first report describing the analysis of a gene encoding an α-glucuronidase, an enzyme essential for the complete breakdown of substituted xylans. A DNA fragment that carries the gene for α-glucuronidase was isolated from chromosomal DNA of the hyperthermophilic bacterium Thermotoga maritima MSB8. The α-glucuronidase gene ( aguA ) was identified and characterized with the aid of nucleotide sequence analysis, deletion experiments and expression studies in Escherichia coli , and the start of the coding region was defined by amino-terminal sequencing of the purified recombinant enzyme. The aguA gene encodes a 674-amino-acid, largely hydrophilic polypeptide with a calculated molecular mass of 78 593 Da. The α-glucuronidase of T. maritima has a novel primary structure with no significant similarity to any other known amino acid sequence. The recombinant enzyme was purified to homogeneity as judged by SDS–PAGE. Gel filtration analysis at low salt concentrations revealed a high apparent molecular mass (<630kDa) for the recombinant enzyme, but the oligomeric structure changed upon variation of the ionic strength or the pH, yielding hexameric and/or dimeric forms which were also enzymatically active. The enzyme hydrolysed 2- O -(4- O -methyl-α- d -glucopyranosyluronic acid)- d -xylobiose (MeGlcAX₂) to xylobiose and 4- O -methylglucuronic acid. The K _m for MeGlcAX₂ was 0.95mM. The pH optimum was 6.3. Maximum activity was measured at 85°C, about 25°C or more above the values reported for all other α-glucuronidases known to date. When incubated at 55–75°C, the enzyme suffered partial inactivation, but thereafter the residual activity remained nearly constant for several days.     

Enrichment of new alkane oxidizing bacterial strains for human drug metabolite production

An extracellular xylanase produced by Streptomyces matensis DW67 was purified from the culture supernatant by ammonium sulfate precipitation, ion exchange and gel filtration chromatography and characterized. The xylanase was purified to 14.5-fold to homogeneity with a recovery yield of 14.1%. The purified xylanase appeared as a single protein band on SDS-PAGE with a molecular mass of 21.2 kDa. However, it had a very low apparent molecular mass of 3.3 kDa as determined by gel filtration chromatography. The N-terminal sequence of first 15 amino acid residues was determined as ATTITTNQTGYDGMY. The optimal temperature and pH for purified xylanase was 65 °C and pH 7.0, respectively. The enzyme was stable within the pH range of 4.5–8.0 and was up to 55 °C. The xylanase showed specific activity towards different xylans and no activity towards other substrates tested. Hydrolysis of birchwood xylan by the xylanase yielded xylobiose and xylotriose as principal products. The enzyme hardly hydrolyzed xylobiose and xylotriose, but it could hydrolyze xylotetraose and xylopentaose to produce mainly xylobiose and xylotriose through transglycosylation. These unique properties of the purified xylanase make this enzyme attractive for biotechnological applications, such as bioblenching in paper and pulp industries, production of xylooligosaccharides. This is the first report of the xylanase from S. matensis.     

Purification and properties of xylanase A from alkali-tolerant Bacillus sp. strain BP-23.

Xylanase A from the recently isolated Bacillus sp. strain BP-23 was purified to homogeneity. The enzyme shows a molecular mass of 32 kDa and an isoelectric point of 9.3. Optimum temperature and pH for xylanase activity were 50 degrees C and 5.5 respectively. Xylanase A was completely inhibited by N-bromosuccinimide. The main products of birchwood xylan hydrolysis were xylotetraose and xylobiose. The enzyme was shown to facilitate chemical bleaching of pulp, generating savings of 38% in terms of chlorine dioxide consumption. The amino-terminal sequence of xylanase A has a conserved sequence of five amino acids found in xylanases from family F.     

Purification and Characterization of Two Endoxylanases from Clostridium acetobutylicum ATCC 824

Two endoxylanases produced by C. acetobutylicum ATCC 824 were purified to homogeneity by column chromatography. Xylanase A, which has a molecular weight of 65,000, hydrolyzed larchwood xylan randomly, yielding xylohexaose, xylopentaose, xylotetraose, xylotriose, and xylobiose as end products. Xylanase B, which has a molecular weight of 29,000, also hydrolyzed xylan randomly, giving xylotriose and xylobiose as end products. Xylanase A hydrolyzed carboxymethyl cellulose with a higher specific activity than xylan. It also exhibited high activity on acid-swollen cellulose. Xylanase B showed practically no activity against either cellulose or carboxymethyl cellulose but was able to hydrolyze lichenan with a specific activity similar to that for xylan. Both xylanases had no aryl-β-xylosidase activity. The smallest oligosaccharides degraded by xylanases A and B were xylohexaose and xylotetraose, respectively. The two xylanases demonstrated similar K_m and V_max values but had different pH optima and isoelectric points. Ouchterlony immunodiffusion tests showed that xylanases A and B lacked antigenic similarity.     

The recombinant xylanase B of Thermotoga maritima is highly xylan specific and produces exclusively xylobiose from xylans, a unique character for industrial applications

The xynB of a hyperthermophilic Eubacterium, Thermotoga maritima MSB8, coding xylanase B (XynB) was previously expressed in E. coli and the recombinant protein was characterized using the synthetic substrates [J. Biosci. Bioeng. 92 (2001) 423]. In this study, the same xylanase B was purified to homogeneity with a recovery yield of about 43% using heat treatment followed by the Ni-NTA affinity chromatography. The specificity of XynB towards different natural substrates was evaluated. XynB was highly specific towards xylans tested but exhibited low activities towards lichenan (19%), gellan gum (7.3%), laminarin (3.4%) and carboxymethylcellulose (CMC, 1.4%). The apparent K_m values of birchwood xylan and soluble oat-spelt xylan was 0.11 and 0.079 mg/ml, respectively. The XynB hydrolyzed xylooligosaccharides to yield predominantly xylobiose (X₂) and a small amount of xylose (X₁), suggesting that XynB was possibly an endo-acting xylanase. Analysis of the products from birchwood xylan degradation confirmed that the enzyme was an endo-xylanase with xylobiose and xylose as the main degradation products. HPLC results showed that hydrolyzed products of birchwood xylan by XynB yielded up to 66% of the total reaction product as xylobiose. These results clearly indicated that xylobiose could be mass-produced efficiently by the recombinant hyperthermostable XynB of T. maritima. Additionally, conversion of xylobiose (50 mM) to xylose was observed, while xylotriose (X₃) and xylotetraose (X₄) were detected in small amounts, indicating that the enzyme converted xylobiose to xylose based on the transglycosylation reaction. The increased binding ability of XynB to Avicel and/or insoluble xylan was also observed indicating the possibilities of roles of surface-aromatic amino acid residues for such action. However, further investigations are required to prove this speculation.     

Biochemical and catalytic properties of an endoxylanase purified from the culture filtrate of Sporotrichum thermophile

An endo-beta-1,4-xylanase (1,4-beta-D-xylan xylanoxydrolase, EC 3.2.1.8) present in culture filtrates of Sporotrichum thermophile ATCC 34628 was purified to homogeneity by Q-Sepharose and Sephacryl S-200 column chromatographies. The enzyme has a molecular mass of 25,000 Da, an isoelectric point of 6.7, and is optimally active at pH 5 and at 70 degrees C. Thin-layer chromatography (TLC) analysis showed that endo-xylanase liberates mainly xylose (Xyl) and xylobiose (Xyl2) from beechwood 4-O-methyl-D-glucuronoxylan, O-acetyl-4-O-methylglucuronoxylan and rhodymenan (a beta-(1-->4)-beta(1-->3)-xylan). Also, the enzyme releases an acidic xylo-oligosaccharide from 4-O-methyl-D-glucuronoxylan, and an isomeric xylotetraose and an isomeric xylopentaose from rhodymenan. Analysis of reaction mixtures by high performance liquid chromatography (HPLC) revealed that the enzyme cleaves preferentially the internal glycosidic bonds of xylooligosaccharides, [1-3H]-xylooligosaccharides and xylan. The enzyme also hydrolyses the 4-methylumbelliferyl glycosides of beta-xylobiose and beta-xylotriose at the second glycosidic bond adjacent to the aglycon. The endoxylanase is not active on pNPX and pNPC. The enzyme mediates a decrease in the viscosity of xylan associated with a release of only small amounts of reducing sugar. The enzyme is irreversibly inhibited by series of omega-epoxyalkyl glycosides of D-xylopyranose. The results suggest that the endoxylanase from S. thermophile has catalytic properties similar to the enzymes belonging to family 11.     

Isolation and characterization of a cellulase-free endo-β-1,4-xylanase produced by an invertebrate-symbiotic bacterium, Cellulosimicrobium sp. HY-13

A xylanolytic bacterium, Cellulosimicrobium sp. HY-13, was isolated from the digestive tract of an earthworm, Eisenia fetida. The purified cellulase-free endo-β-1,4-xylanase (XylK) produced by strain HY-13 was found to contain an N-terminal amino acid sequence of APSTLEAAAE and to have a relative molecular mass of 36 kDa. It was most active at pH 6.0 and 55 °C and had V_max and K_m values toward oat spelt xylan of 4067 IU/mg and 2.78 mg/ml, respectively. XylK primarily degraded xylan to a series of xylooligosaccharides composed of xylobiose to xylotetraose, but it could not further hydrolyze xylobiose to xylose. The results of the present study suggest that the relatively highly active XylK lacking exo-xylanolytic activity is a promising candidate for the efficient production of non-digestible xylooligosaccharides that may have beneficial effects to gastrointestinal health via promotion of the growth of probiotics.     

Production, Purification, and Characterization of beta-(1-4)-Endoxylanase of Streptomyces roseiscleroticus

Twelve species of Streptomyces that formerly belonged to the genus Chainia were screened for the production of xylanase and cellulase. One species, Streptomyces roseiscleroticus (Chainia rosea) NRRL B-11019, produced up to 16.2 IU of xylanase per ml in 48 h. A xylanase from S. roseiscleroticus was purified and characterized. The enzyme was a debranching beta-(1-4)-endoxylanase showing high activity on xylan but essentially no activity against acid-swollen (Walseth) cellulose. It had a very low apparent molecular weight of 5,500 by native gel filtration, but its denatured molecular weight was 22,600 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It had an isoelectric point of 9.5. The pH and temperature optima for hydrolysis of arabinoxylan were 6.5 to 7.0 and 60 degrees C, respectively, and more than 75% of the optimum enzyme activity was retained at pH 8.0. The xylanase had a K(m) of 7.9 mg/ml and an apparent V(max) of 305 mumol . min . mg of protein. The hydrolysis rate was linear for xylan concentrations of less than 4 mg/ml, but significant inhibition was observed at xylan concentrations of more than 10 mg/ml. The predominant products of arabinoxylan hydrolysis included arabinose, xylobiose, and xylotriose.     

Production, Purification, and Characterization of β-(1-4)-Endoxylanase of Streptomyces roseiscleroticus

Twelve species of Streptomyces that formerly belonged to the genus Chainia were screened for the production of xylanase and cellulase. One species, Streptomyces roseiscleroticus (Chainia rosea) NRRL B-11019, produced up to 16.2 IU of xylanase per ml in 48 h. A xylanase from S. roseiscleroticus was purified and characterized. The enzyme was a debranching β-(1-4)-endoxylanase showing high activity on xylan but essentially no activity against acid-swollen (Walseth) cellulose. It had a very low apparent molecular weight of 5,500 by native gel filtration, but its denatured molecular weight was 22,600 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It had an isoelectric point of 9.5. The pH and temperature optima for hydrolysis of arabinoxylan were 6.5 to 7.0 and 60°C, respectively, and more than 75% of the optimum enzyme activity was retained at pH 8.0. The xylanase had a K_m of 7.9 mg/ml and an apparent V_max of 305 μmol · min^-1 · mg of protein^-1. The hydrolysis rate was linear for xylan concentrations of less than 4 mg/ml, but significant inhibition was observed at xylan concentrations of more than 10 mg/ml. The predominant products of arabinoxylan hydrolysis included arabinose, xylobiose, and xylotriose.     

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.     

Purification and properties of the membrane-associated coenzyme F420-reducing hydrogenase from Methanobacterium formicicum.

The membrane-associated coenzyme F420-reducing hydrogenase of Methanobacterium formicicum was purified 87-fold to electrophoretic homogeneity. The enzyme contained alpha, beta, and gamma subunits (molecular weights of 43,000, 36,700, and 28,800, respectively) and formed aggregates (molecular weight, 1,020,000) of a coenzyme F420-active alpha 1 beta 1 gamma 1 trimer (molecular weight, 109,000). The hydrogenase contained 1 mol of flavin adenine dinucleotide (FAD), 1 mol of nickel, 12 to 14 mol of iron, and 11 mol of acid-labile sulfide per mol of the 109,000-molecular-weight species, but no selenium. The isoelectric point was 5.6. The amino acid sequence I-N3-P-N2-R-N1-EGH-N6-V (where N is any amino acid) was conserved in the N-termini of the alpha subunits of the F420-hydrogenases from M. formicicum and Methanobacterium thermoautotrophicum and of the largest subunits of nickel-containing hydrogenases from Desulfovibrio baculatus, Desulfovibrio gigas, and Rhodobacter capsulatus. The purified F420-hydrogenase required reductive reactivation before assay. FAD dissociated from the enzyme during reactivation unless potassium salts were present, yielding deflavoenzyme that was unable to reduce coenzyme F420. Maximal coenzyme F420-reducing activity was obtained at 55 degrees C and pH 7.0 to 7.5, and with 0.2 to 0.8 M KCl in the reaction mixture. The enzyme catalyzed H2 production at a rate threefold lower than that for H2 uptake and reduced coenzyme F420, methyl viologen, flavins, and 7,8-didemethyl-8-hydroxy-5-deazariboflavin. Specific antiserum inhibited the coenzyme F420-dependent but not the methyl viologen-dependent activity of the purified enzyme.     

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+).