Catalytic properties of the endoxylanase I from Thermoascus aurantiacus

Endo-β-1,4-xylanase I previously purified from Thermoascus aurantiacus solid state culture was further characterized. The enzyme had a molecular weight of 33 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and 31 kDa by gel filtration. Thin layer chromatography (TLC) analysis showed that endoxylanase liberates aldotetrauronic acid MeGlcA-1,2-Xylβ-1,4-Xylβ-1,4-Xyl as the shortest acidic fragment from glucuronoxylan and an isomeric xylotriose (Xyl₃) of the structure Xylβ1-3Xylβ1-4Xyl from rhodymenan. The enzyme performed ideally on O-acetyl-4-O-methylglucuronoxylan, liberating large amounts of short acetylated and non-acetylated fragments. Also, the enzyme was capable to hydrolyse arabinoxylan to arabinose (Arab), xylose (Xyl) and xylobiose (Xyl₂). The enzyme degraded pNPX (4-nitrophenyl β- -xylopyranoside) by a complex reaction pathway that involved both hydrolysis and glycosyl transfer reactions. The enzyme tolerates the replacement of β-xylopyranosyl units in several artificial substrates by β-glucopyranosyl, - -arabinopyranosyl and - -arabinofuranosyl units and was active on pNPC (4-nitrophenyl β- -cellobioside), pNP-Arap (4-nitrophenyl - -arabinopyranoside) and pNPAraf (4-nitrophenyl - -arabinofuranoside). The enzyme also hydrolysed the 4-methylumbelliferyl glycosides of β- -xylobiose and β- -xylotriose at the agluconic linkage. The results suggested that the xylanase I from T. aurantiacus has catalytic properties similar to those belonging to family 10.     

Purification and characterization of two minor endo-β-1,4-xylanases of Schizophyllum commune

Two minor extracellular endo-β-1,4-xylanases (XynB and XynC, EC 3.2.1.8) were purified from the culture filtrate of Schizophyllum commune grown on cellulose. The molecular mass of enzymes was estimated to be 30.5 kDa for XynB and 30 kDa for XynC according to SDS-PAGE. Both enzymes were acidic, with pI value 2.8 for XynB and 3.6 for XynC. The highest activities were achieved at 50 °C and pH 5.5 and enzymes were stable up to 40 °C in the pH range 5–7. A comparison of hydrolysis products of glucuronoxylan, rhodymenan and acetylxylan showed different mode of action of all three xylanases of S. commune. Known XynA generated products typical for family 11 of glycoside hydrolase – aldopentaouronic acid from glucuronoxylan and isomeric xylotetraose from rhodymenan. XynB released fragments by one xylopyranosyl unit shorter – aldotetraouronic acid MeGlcA1-2Xylβ1-4Xylβ1-4Xyl from glucuronoxylan and isomeric xylotriose from rhodymenan, products usually generated by xylanases from glycoside hydrolase family 10. XynC liberated aldotetraouronic acid Xylβ-1,4-(MeGlcA-1,2-)Xylβ-1,4-Xyl with glucuronoyl unit attached to the middle xylopyranosyl unit from glucuronoxylan and isomeric xylotetraose from rhodymenan. XynC was also able to release xylose from the reducing end of aldotetraouronic acid MeGlcA1-2Xylβ1-4Xylβ1-4Xyl.     

Purification and properties of a low molecular weight 1,4-beta-d-glucan glucohydrolase having one active site for carboxymethyl cellulose and xylan from an alkalothermophilic Thermomonospora sp

A low molecular weight 1,4-beta-D-glucan glucohydrolase from an extracellular culture filtrate of Thermomonospora sp. was purified to homogeneity. The molecular weight of the purified enzyme was 14.2 kDa by MALDI-TOF analysis and is in agreement with SDS-PAGE and gel filtration chromatography. The purified enzyme exhibited both endocarboxymethyl cellulase and endoxylanase activities. A kinetic method was employed to study the active site of the enzyme that hydrolyzes both carboxymethyl cellulose and xylan. The experimental data coincide well with the theoretical values calculated for the case of a single active site. Conformation and microenvironment at the active site was probed with fluorescent chemo-affinity labeling using o-phthalaldehyde as the chemical initiator. Formation of isoindole derivative resulted in complete inactivation of the enzyme to hydrolyze both xylan and CMC as judged by fluorescence studies corroborating a single active site for the hydrolysis of xylan and CMC.     

Structure elucidation of arabinoxylan isomers by normal phase HPLC-MALDI-TOF/TOF-MS/MS

Normal phase-high performance liquid chromatography (NP-HPLC) coupled to matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight (MALDI-TOF/TOF) tandem mass spectrometry is evaluated for the detailed structural characterization of various isomers of arabinoxylan (AX) oligosaccharides produced from endo-beta-(1-->4)-xylanase (endoxylanase) digestion of wheat AX. The fragmentation characteristics of these oligosaccharides upon MALDI-TOF/TOF high-energy collision induced dissociation (CID) were investigated using purified AX oligosaccharide standards labeled at the reducing end with 2-aminobenzoic acid (2-AA). A variety of cross-ring cleavages and 'elimination' ions in the fragment ion spectra provided extensive structural information, including Araf substitution patterns along the xylan backbone and comprehensive linkage assignment. The off-line coupling of this MALDI-CID technique to capillary normal phase HPLC enabled the separation and identification of isomeric oligosaccharides (DP 4-8) produced by endoxylanase digestion of AX. Furthermore, this technique was used to characterize structurally different isomeric AX oligosaccharides produced by endoxylanase enzymes with different substrate specificities.     

Purification and characterization of Thermobifida fusca xylanase 10B

Thermobifida fusca grows well on cellulose and xylan, and produces a number of cellulases and xylanases. The gene encoding a previously unstudied endoxylanase, xyl10B, was overexpressed in E. coli, and the protein was purified and characterized. Mature Xyl10B is a 43-kDa glycohydrolase with a short basic domain at the C-terminus. It has moderate thermostability, maintaining 50% of its activity after incubation for 16 h at 62 degrees C, and is most active between pH 5 and 8. Xyl10B is produced by growth of T. fusca on xylan or Solka Floc but not on pure cellulose. Mass spectroscopic analysis showed that Xyl10B produces xylobiose as the major product from birchwood and oat spelts xylan and that its hydrolysis products differ from those of T. fusca Xyl11A. Xyl10B hydrolyzes various p-nitrophenyl-sugars, including p-nitrophenyl alpha-D-arabinofuranoside, p-nitrophenyl-beta-D-xylobioside, p-nitrophenyl-beta-D-xyloside, and p-nitrophenyl-beta-D-cellobioside. Xyl11A has higher activity on xylan substrates, but Xyl10B produced more reducing sugars from corn fiber than did Xyl11A.     

Enzymatic properties of a neutral endo-1,3(4)-beta-xylanase Xyl II from Bacillus subtilis

A Bacillus sp. CCMI 966, characterised as Bacillus subtilis, has a duplication time of about 24 min. It produces at least two extracellular xylanases, Xyl I and Xyl II. The extracellular xylanase activity seems to be strongly correlated with the biomass growth profile. The Xyl II isoenzyme was purified by ammonium sulphate precipitation and anionic exchange chromatography, with a purification factor of 8.3. The molecular weight of the isoenzyme was estimated by SDS-PAGE revealing that Xyl II is a multimeric enzyme with a catalytic subunit of about 20 kDa. Under non-denaturing conditions, a molecular weight of about 340 kDa was obtained by native PAGE gel and of 20 kDa by gel filtration chromatography. The enzyme showed an optimum pH and temperature of 6.0 at 60 degrees C. Xyl II was stable at 40 degrees C for 180 min at pH 6.0. The specificity of Xyl II for different substrates was evaluated. Xyl II presents a higher affinity towards OSX, with a K(m) of 1.56 g l(-1) and showed the ability to hydrolyse laminarin, with a K(m) of 1.02 g l(-1). Xylotetraose is the main product of xylan degradation. The Xyl II ability for binding to cellulose and/or xylan was also studied.     

Purification and characteristic of endo-(1--4)-beta-xylanase from Geotrichum candidum 3C

A method of purification of endo-(1-->4)-beta-xylanase (endoxylanase; EC 3.2.1.8) from the culture liquid of Geotrichum candidum 3C, grown for three days, is described. The enzyme purified 23-fold had a specific activity of 32.6 U per mg protein (yield, 14.4%). Endoxylanase was shown to be homogeneous by SDS-PAGE (molecular weight, 60 to 67 kDa). With carboxymethyl xylan as substrate, the optimum activity (determined viscosimetrically) was recorded at pH 4.0 (pI 3.4). The enzyme retained stability at pH 3.0-4.5 and 30-45 degrees C for 1 h. With xylan from beach wood, the hydrolytic activity of the enzyme (ability to saccharify the substrate) was maximum at 50 degrees C. In 72 h of exposure to 0.2 mg/ml endoxylanase, the extent of saccharification of xylans from birch wood, rye grain, and wheat straw amounted to 10, 12, and 7.7%, respectively. At 0.4 mg/ml, the extent of saccharification of birch wood xylan was as high as 20%. In the case of birch wood xylan, the initial hydrolysis products were xylooligosaccharides with degrees of polymerization in excess of four; the end products were represented by xylobiose, xylotriose, xylose, and acid xylooligosaccharides.     

Improvement of fungal arabinofuranosidase thermal stability by reversible immobilization

A gene encoding α-l-arabinofuranosidase (abfA) from Aspergillus niveus was identified, cloned, and successfully expressed in Aspergillus nidulans. Based on amino acid sequence comparison, the 88.6 kDa enzyme could be assigned to the GH family 51. The characterization of the purified recombinant AbfA revealed that the enzyme was active at a limited pH range (pH 4.0–5.0) and an optimum temperature of 70 °C. The AbfA was able to hydrolyze arabinoxylan, xylan from birchwood, debranched arabinan, and 4-nitrophenyl arabinofuranoside. Synergistic reactions using both AbfA and endoxylanase were also assessed. The highest degree of synergy was obtained after the sequential treatment of the substrate with endoxylanase, followed by AbfA, which was observed to release noticeably more reducing sugars than that of either enzyme acting individually. The immobilization of AbfA was performed via ionic adsorption onto various supports: agarose activated by polyethyleneimine polymers, cyanogen bromide activated Sepharose, DEAE-Sepharose, and Sepharose-Q. The Sepharose-Q derivative remained fully active at pH 5 after 360 min at 60 °C, whereas the free AbfA was inactivated after 60 min. A synergistic effect of arabinoxylan hydrolysis by AbfA immobilized in Sepharose-Q and endoxylanase immobilized in glyoxyl agarose was also observed. The stabilization of arabinofuranosidases using immobilization tools is a novel and interesting topic.     

A xylan hydrolase gene cluster in Prevotella ruminicola B(1)4: sequence relationships, synergistic interactions, and oxygen sensitivity of a novel enzyme with exoxylanase and beta-(1,4)-xylosidase activities.

Two genes concerned with xylan degradation were found to be closely linked in the ruminal anaerobe Prevotella ruminicola B(1)4, being separated by an intergenic region of 75 nucleotides. xynA is shown to encode a family F endoxylanase of 369 amino acids, including a putative amino-terminal signal peptide. xynB encodes an enzyme of 319 amino acids, with no obvious signal peptide, that shows 68% amino acid identity with the xsa product of Bacteroides ovatus and 31% amino acid identity with a beta-xylosidase from Clostridium stercorarium; together, these three enzymes define a new family of beta-(1,4)-glycosidases. The activity of the cloned P. ruminicola xynB gene product, but not that of the xynA gene product, shows considerable sensitivity to oxygen. Studied under anaerobic conditions, the XynB enzyme was found to act as an exoxylanase, releasing xylose from substrates including xylobiose, xylopentaose, and birch wood xylan, but was relatively inactive against oat spelt xylan. A high degree of synergy (up to 10-fold stimulation) was found with respect to the release of reducing sugars from oat spelt xylan when XynB was combined with the XynA endoxylanase from P. ruminicola B(1)4 or with endoxylanases from the cellulolytic rumen anaerobe Ruminococcus flavefaciens 17. Pretreatment with a fungal arabinofuranosidase also stimulated reducing-sugar release from xylans by XynB. In P. ruminicola the XynA and XynB enzymes may act sequentially in the breakdown of xylan.     

A Simple Determination Method for Herbicides,NIP and CNP in Soil Using Single Ion Monitoring Mass Spectrometry

A purified extracellular endo β-1,3-xylanase (EC 3.2.1.32) from an isolated strain, Aspergillus terreus A-07, was found to hydrolyze 1,3-xylosyl linkages only. When rhodymenan (β-1,4 and β-1.3-linked xylan) was hydrolyzed by β-1,3-xylanase (EF-6), four β-1,4-linked xylooligosaccharide fractions were produced. The main product was β-1,4-xylotriose, with trace amounts of other β-1,4-linked xylooligosaccharides. Successive degradation by β-l,4-xylosidase of the β,4-xylooligosaccharides that were produced from hydrolysis of β-1,3-xylanase on rhodymenan yielded only xylose as the final product.

We compared the action pattern of this enzyme with that of an extracellular endo β-l,4-xylanase (EC 3.2.1.8) of Streptomyces. From a mixture of products of β-1,4-xylanase hydrolysis on rhodymenan, an isomeric xylotriose was isolated by charcoal chromatography after treating with β-1.4-xylosidase. The structure of this isomeric xylotriose was elucidated by methylation analysis and its susceptibility to β-1,4-xylanase, β-1,3-xylanase, and β-1,4-xylosidase. The obtained isomeric xylotriose was identified as 3-O-β-xylopyranosyl-4-O-β-D-xylopyranosyl-D-xylose (X1→3X1→4X). It has a melting point of 224~225°C and ${\left[\alpha \right]}_{\text{D}}^{20}$(c = 1, H₂O)= —46°.     

Xylanases from Cellulomonas flavigena: purification and characterization

Three xylanases (Xyl1, Xyl2 and Xyl3) were purified and characterized from the culture supernatant of Cellulomonas flavigena grown on sugar cane bagasse. The enzymes were purified by affinity chromatography and gel filtration and had masses of 63 kDa, 17 kDa and 35 kDa, respectively, as measured by SDS-PAGE. All enzymes were active against 4-O-methyl-D-glucuronoxylan and xylan but had no cellulase activity with CM-cellulose, an important characteristics in biobleaching processes. © Rapid Science Ltd. 1998     

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 Cooperative Activity of Enzymes Constituting the Xylan-Degrading System of Thermomonospora fusca

The thermophilic actinomycete Thermomonospora fusca produced endoxylanase, α-arabinofuranosidase, β-xylosidase, and acetyl esterase activities maximally during growth on xylan. Growth yields on glucose, xylose, or arabinose were comparable, but production of endoxylanase and β-xylosidase was not induced on these substrates. The crude xylanase activity was thermostable and relatively resistant to end product inhibition by xylobiose and xylan hydrolysis products. Six proteins with xylanase activity were identified by zymogram analysis of isoelectric focusing gels, but only a 32-kDa protein exhibiting three isomeric forms could be purified by fast protein liquid chromatography. Endoglucanases were also identified in carboxymethylcellulose-grown cultures, and their distinction from endoxylanases was confirmed. α-Arabinofuranosidase activity was due to a single dimeric protein of 92 kDa, which was particularly resistant to end product inhibition by arabinose. Three bands of acetyl esterase activity were detected by zymogram analysis, and there was evidence that these mainly consisted of an intracellular 80-kDa protein secreted to yield active 40-kDa subunits in the culture supernatant. The acetyl esterases were found to be responsible for acetyl xylan esterase activity in T. fusca, in contrast to the distinction proposed in some other systems. The addition of purified βxylosidase to endoxylanase increased the hydrolysis of xylan, probably by relieving end product inhibition. The enhanced saccharification of wheat straw caused by the addition of purified α-arabinofuranosidase to T. fusca endoxylanase suggested a truly synergistic relationship, in agreement with proposals that arabinose side groups on the xylan chain participate in cross-linking within the plant cell wall structure.     

Purification and biochemical characterization of an endoxylanase from Aspergillus versicolor

An endoxylanase (β-1,4-xylan xylanohydrolase, EC 3.2.1.8) was purified from the culture filtrate of a strain of Aspergillus versicolor grown on oat wheat. The enzyme was purified to homogeneity by chromatography on DEAE-cellulose and Sephadex G-75. The purified enzyme was a monomer of molecular mass estimated to be 19 kDa by SDS-PAGE and gel filtration. The enzyme was glycoprotein with 71% carbohydrate content and exhibited a pI of 5.4. The purified xylanase was specific for xylan hydrolysis. The enzyme had a K _m of 6.5 mg ml⁻¹ and a V _max of 1440 U (mg protein)⁻¹.     

An enzyme activity capable of endotransglycosylation of heteroxylan polysaccharides is present in plant primary cell walls

Heteroxylans in the plant cell wall have been proposed to have a role analogous to that of xyloglucans or heteromannans, forming growth-restraining networks by interlocking cellulose microfibrils. A xylan endotransglycosylase has been identified that can transglycosylate heteroxylan polysaccharides in the presence of xylan-derived oligosaccharides. High activity was detected in ripe fruit of papaya (Carica papaya), but activity was also found in a range of other fruits, imbibed seeds and rapidly growing seedlings of cereals. Xylan endotransglycosylase from ripe papaya fruit used a range of heteroxylans, such as wheat arabinoxylan, birchwood glucuronoxylan and various heteroxylans from dicotyledonous primary cell walls purified from tomato and papaya fruit, as donor molecules. As acceptor molecules, the enzyme preferentially used xylopentaitol over xylohexaitol or shorter-length acceptors. Xylan endotransglycosylase was active over a broad pH range and could perform transglycosylation reactions up to 55 °C. Xylan endotransglycosylase activity was purified from ripe papaya fruit by ultrafiltration and cation exchange chromatography. Highest endotransglycosylase activity was identified in fractions that also contained high xylan hydrolase activity and correlated with the presence of the endoxylanase CpaEXY1. Recombinant CpaEXY1 protein transiently over-expressed in Nicotiana benthamiana leaves showed both endoxylanase and xylan endotransglycosylase activities in vitro, suggesting that CpaEXY1 is a single enzyme with dual activity in planta. Purified native CpaEXY1 showed two- to fourfold higher endoxylanase than endotransglycosylase activity, suggesting that CpaEXY1 may act primarily as a hydrolase. We propose that xylan endotransglycosylase activity (like xyloglucan and mannan endotransglycosylase activities) could be involved in remodelling or re-arrangement of heteroxylans of the cellulose-non-cellulosic cell wall framework.     

Purification of an isoflavonoid 7-O-beta-apiosyl-glucoside beta-glycosidase and its substrates from Dalbergia nigrescens Kurz

A beta-glycosidase was purified from the seeds of Dalbergia nigescens Kurz based on its ability to hydrolyse p-nitrophenyl beta-glucoside and beta-fucoside. This enzyme did not hydrolyze various glycosidic substrates efficiently, so it was used to identify its own natural substrates. Two substrates were identified, isolated and their structures determined as: compound 1, dalpatein 7-O-beta-D-apiofuranosyl-(1-->6)-beta-D-glucopyranoside and compound 2, 6,2',4',5'-tetramethoxy-7-hydroxy-7-O-beta-D-apiofuranosyl-(1-->6)-beta-D-glucopyranoside (dalnigrein7-O-beta-D-apiofuranosyl-(1-->6)-beta-D-glucopyranoside). The beta-glycosidase removes the sugar from these glycosides as a disaccharide, despite its initial identification as a beta-glucosidase and beta-fucosidase.     

Hydrolysis of wheat bran and straw by an endoxylanase: production and structural characterization of cinnamoyl-oligosaccharides.

Hydrolysis of wheat bran and wheat straw by a 20.7 kDa thermostable endoxylanase released 35 and 18% of the cell-wall xylan content, respectively. Separation of the cinnamoyl-oligosaccharides (accounting for 6%) from the bulk of total oligosaccharides was achieved by specific anion-exchange chromatography. The cinnamoyl-oligosaccharides were further purified by preparative paper chromatography (PPC) and their molecular weight was determined by MALDI-TOF mass spectrometry. The partially purified hydrolysis end-products contained from 4 to 16 and from 4 to 12 pentose residues for wheat bran and straw, respectively, and only one cinnamic acid per molecule. The primary structure of the new feruloyl arabinoxylopentasaccharide from wheat bran hydrolysis, which has been determined using 2D NMR spectroscopy, is O-beta-D-xylopyranosyl-(1-->4)-O-[5-O- (feruloyl)-alpha-L-arabinofuranosyl-(1-->3)]-O-beta-D-xylopyranosy l-(1-->4) -O-beta-D-xylopyranosyl-(1-->4)-D-xylopyranose.     

Hydrolysis of Nothogenia erinacea xylan by xylanases from families 10 and 11

The structures of several enzymatic hydrolysis products of Nothogenia erinacea seaweed xylan, a linear homopolymer with mixed beta-(1-->3)/beta-(1-->4) linkages, were analysed by physicochemical and biochemical techniques. With the glycoside hydrolase family 10 beta-(1-->4)-xylanase from Cryptococcus adeliae, hydrolysis proceeds to a final mixture of products containing a mixed linkage-type triose as a major compound, whereas with the family 11 xylanase from Thermomyces lanuginosus this is a mixed linkage tetraose. The Cryptococcus xylanase is shown to be capable of also catalysing the hydrolysis of beta-(1-->3) linkages, that is this of a mixed type tetraose intermediary formed, in accordance with the broader substrate specificity of family 10 enzymes. From a partial degradation experiment with the T. lanuginosus xylanase, a series of higher mixed oligosaccharides were isolated and identified. The observed oligosaccharide intermediates and splicing pattern indicate an irregular beta-(1-->3)/beta-(1-->4) linkage distribution within the linear d-xylose polymer. Similar results were obtained with rhodymenan, the seaweed xylan from Palmares palmata.     

Antimicrobial activity of acidic xylo-oligosaccharides produced by family 10 and 11 endoxylanases

Acidic oligosaccharides were obtained from birchwood xylan by treatment with a Thermoascus aurantiacus family 10 and a Sporotrichum thermophile family 11 endoxylanases. The main difference between the products liberated by xylanases of family 10 and 11 concerned the length of the products containing 4-O-methyl-D-glucuronic acid. The xylanase from T. aurantiacus liberate from glucuronoxylan an aldotetrauronic acid as the shortest acidic fragment in contrast with the enzyme from S. thermophile, which liberated an aldopentauronic acid. Acidic xylooligosaccharides were separated from the hydrolysate by anion-exchange and size-exclusion chromatography (SEC) and the primary structure was determined by 13C NMR spectroscopy. The acidic xylo-oligosaccharides were tested against three Gram-positive and three Gram-negative aerobically grown bacteria, as well as against Helicobacter pylori. Aldopentauronic acid was proved more active against the Gram-positive bacteria and against H. pylori.