Oat Spelt Xylan Research Articles

Purification and Properties of a Thermostable Xylanase GH 11 from Penicillium occitanis Pol6

An extracellular, endo-β-1,4-xylanase was purified to homogeneity from the culture filtrate of the filamentous fungus Penicillium occitanis Pol6, grown on oat spelt xylan. The purified enzyme (PoXyn2) showed a single band on SDS-PAGE with an apparent molecular weight of 30 kDa. The xylanase activity was optimal at pH 3.0 and 65 °C. The specific activity measured for oat spelt xylan was 2,368 U mg(-1). The apparent K(m) and V(max) values were 8.33 mg ml(-1) and 58.82 μmol min(-1) ml(-1), respectively, as measured on oat spelt xylan. Thin-layer chromatography experiments revealed that purified PoXyn2 degrades xylan in an endo-fashion releasing xylobiose as main end product. The genomic DNA and cDNA encoding this protein were cloned and sequenced. This PoXyn2 presents an open reading frame of 962 bp, not interrupted by any introns and encoding for a mature protein of 320 amino acids and 29.88 kDa.

Structural modeling and further improvement in pH stability and activity of a highly-active xylanase from an uncultured rumen fungus

Rumen fungi are a rich source of enzymes degrading lignocelluloses. XynR8 is a glycosyl hydrolase family 11 xylanase previously cloned from unpurified rumen fungal cultures. Phylogenetic analysis suggested that xynR8 was obtained from a Neocallimastix species. Recombinant XynR8 expressed in Escherichia coli was highly active and stable between pH 3.0 and 11.0, and displayed a Vmax of 66,672μmolmin−1mg−1, a kcat of 38,975s−1, and a Km of 11.20mg/mL towards soluble oat spelt xylan. Based on molecular modeling, residues N41 and N58, important in stabilizing two loops and the structure of XynR8, were mutated to D. Both mutant enzymes showed higher tolerance to pH 2.0. The Vmax, kcat and Km of the N41D and N58D mutant enzymes were 79,645μmolmin−1mg−1, 46,493s−1, 29.29mg/mL, and 96,689μmolmin−1mg−1, 56,503s−1, and 21.24mg/mL, respectively. Thus, they are good candidates for application, including biofuel production.

Characterization of Xyn30A and Axh43A of Bacillus licheniformis SVD1 identified by its genomic analysis

The genome sequence of Bacillus licheniformis SVD1, that produces a cellulolytic and hemi-cellulolytic multienzyme complex, was partially determined, indicating that the glycoside hydrolase system of this strain is highly similar to that of B. licheniformis ATCC14580. All of the fifty-six genes encoding glycoside hydrolases identified in B. licheniformis ATCC14580 were conserved in strain SVD1. In addition, two new genes, xyn30A and axh43A, were identified in the B. licheniformis SVD1 genome. The xyn30A gene was highly similar to Bacillus subtilis subsp. subtilis 168 xynC encoding for a glucuronoarabinoxylan endo-1,4-β-xylanase. Xyn30A, produced by a recombinant Escherichia coli, had high activity toward 4-O-methyl-d-glucurono-d-xylan but showed definite activity toward oat-spelt xylan and unsubstituted xylooligosaccharides. Recombinant Axh43A, consisting of a family-43 catalytic module of the glycoside hydrolases and a family-6 carbohydrate-binding module (CBM), was an arabinoxylan arabinofuranohydrolase (α-l-arabinofuranosidase) classified as AXH-m23 and capable of releasing arabinosyl residues, which are linked to the C-2 or C-3 position of singly substituted xylose residues in arabinoxylan or arabinoxylan oligomers. The isolated CBM polypeptide had an affinity for soluble and insoluble xylans and removal of the CBM from Axh43A abolished the catalytic activity of the enzyme, indicating that the CBM plays an essential role in hydrolysis of arabinoxylan.

Mapping the polysaccharide degradation potential of Aspergillus niger

BackgroundThe degradation of plant materials by enzymes is an industry of increasing importance. For sustainable production of second generation biofuels and other products of industrial biotechnology, efficient degradation of non-edible plant polysaccharides such as hemicellulose is required. For each type of hemicellulose, a complex mixture of enzymes is required for complete conversion to fermentable monosaccharides. In plant-biomass degrading fungi, these enzymes are regulated and released by complex regulatory structures. In this study, we present a methodology for evaluating the potential of a given fungus for polysaccharide degradation.ResultsThrough the compilation of information from 203 articles, we have systematized knowledge on the structure and degradation of 16 major types of plant polysaccharides to form a graphical overview. As a case example, we have combined this with a list of 188 genes coding for carbohydrate-active enzymes from Aspergillus niger, thus forming an analysis framework, which can be queried. Combination of this information network with gene expression analysis on mono- and polysaccharide substrates has allowed elucidation of concerted gene expression from this organism. One such example is the identification of a full set of extracellular polysaccharide-acting genes for the degradation of oat spelt xylan.ConclusionsThe mapping of plant polysaccharide structures along with the corresponding enzymatic activities is a powerful framework for expression analysis of carbohydrate-active enzymes. Applying this network-based approach, we provide the first genome-scale characterization of all genes coding for carbohydrate-active enzymes identified in A. niger.

Xylans inhibit enzymatic hydrolysis of lignocellulosic materials by cellulases

Hemicelluloses have been found to be physical barriers in the hydrolysis of cellulose, and prevent the access of enzymes to cellulose surface. In addition, soluble hemicelluloses may strongly inhibit the cellulase activity. In this work, birchwood xylan clearly inhibited the enzymatic hydrolysis of wheat straw, Avicel and nanocellulose by cellulases. Hydrolysis efficiencies of cellobiohydrolase I (CBHI, from Thermoascus aurantiacus), cellobiohydrolase II (CBHII, from Trichoderma reesei) and endoglucanase II (from T. aurantiacus) were clearly inhibited by birchwood xylan, respectively. The strongest inhibitory effect of birchwood xylan was observed on the hydrolysis of Avicel by CBHI and CBHII, as a dramatically decreased formation of the main product, cellobiose. After additions of soluble and insoluble oat spelt xylan, cleaved cellobiose units by CBHI from cellulose chain decreased from 8 to 4 and 6, respectively. The results in this work demonstrated that xylans clearly inhibited the hydrolysis efficiencies of both endoglucanase and cellobiohydrolase.

양송이 수확 후 배지로부터 분리한 Bacillus subtilis AB-55가 생산하는 xylanase의 특성

A bacterium AB-55, isolated from waste mushroom bed of Agaricus bisporus in Sukseong-myeon, Buyeo-gun, Chungcheongnam-do, Korea, was screened onto xylan agar congo-red plate by the xylanolysis method and was used to produce an xylanase in shaker buffle flask cultures containing oat spelt xylans. The phylogenetic analysis using 16S rRNA gene sequence data showed that the strain AB-55 had the highest homology (99.0%) with Bacillus subtilis and it was named as Bacillus subtilis AB-55. A xylanase was purified by ammonium sulfate precipitation (50~80%), gel filtration on sephacryl S-300, and ion exchange chromatography on DEAE sepharose FF. The molecular weight of the xylanase was estimated as 44 kDa by SDS-PAGE. Optimal pH and temperature for the xylanase activity was pH 7 and <TEX>$50^{\circ}C$</TEX>, respectively. N-terminal amino acid sequence of the enzyme was identified as Ser-Ala-Val-Lys-His-Gly-Ala-Ile-Val-Phe. The substrate specificity of the enzyme exhibited that it hydrolyzed efficiently oat spelt xylan as well as beechwood xylan, but showed no activity against Avicel and carboxymethyl clellulose (CMC). The enzyme activity was enhanced by <TEX>$Fe^{2+}$</TEX> and <TEX>$Mn^{2+}$</TEX> whereas was entirely inhibited by <TEX>$Hg^+$</TEX>.

Biochemical characterization, cloning and molecular modeling of a detergent and organic solvent-stable family 11 xylanase from the newly isolated Aspergillus niger US368 strain

New β-1,4-d-xylan xylanohydrolase (XAn11) belonging to the xylanase 11 family was purified to homogeneity from a newly soil-isolated Aspergillus niger US368 strain. The pure xylanase is a glycosylated monomer having a molecular mass of about 26kDa. The N-terminal sequence of the purified enzyme was determined and compared to some Aspergillus xylanases N-terminal ones. The gene encoding the XAn11 was cloned and sequenced.The maximal xylanase activity was obtained at pH 5.0 and 55°C. The XAn11 was found to be stable in a wide range of pH (3–9) and in presence of some detergents and organic solvents. A specific activity of about 805.6U/mg or 334U/mg was measured using birchwood xylan or oatspelt xylan as substrate, respectively. A structural explanation of the difference between experimental and theoretical molecular mass as well as the stability of the enzyme against acidic pH was proposed by molecular modeling.

Comparative study for the kinetics of extracellular xylanases from Trichoderma harzianum and Chaetomium thermophilum

Xylanases assume special importance in the paper and pulp industry as they replace toxic chemicals such as chlorine and chlorine dioxide for developing eco-friendly processes. This study evaluated xylanases produced by two fungi, the mesophilic fungus Trichoderma harzianum and a thermophilic fungus Chaetomium thermophilum . Among the polymeric substrates studied for xylanase production by both the fungi, birch wood xylan was found to be the best inducer of xylanases. Xylanases induction was subject to glucose repression. Partially purified xylanases preparation from T. harzianum and C. thermophilum exhibited optimal activities at pH 5 and 6 and at 60oC and 70oC, respectively. The apparent K m and V max values for the partially purified xylanase from T. harzianum using oat spelt xylan as a substrate were 4.8 mg mL -1 and 0.526 µmol min -1 mg -1 , respectively. Whereas values of the partially purified xylanase from C. thermophilum were 2.96 mg mL -1 and 0.25 µmol min -1 mg -1 , respectively. These findings in this study have great implications for the future applications of xylanases.

A typical endo-xylanase from Streptomyces rameus L2001 and its unique characteristics in xylooligosaccharide production

The activity of the extracellular xylanase produced by Streptomyces rameus L2001 against different xylans and xylooligosaccharides (XOS) was investigated. The main products of hydrolysis of birchwood xylan and oat-spelt xylan by the S. rameus L2001 xylanase were xylobiose (X2) and xylotriose (X3), suggesting that this is an endo-acting xylanase. This was confirmed by analysis of XOS degradation products. The enzyme hardly hydrolyzed X2 and X3, but hydrolyzed xylotetraose (X4) and xylopentaose (X5) producing mainly X2 and X3 through transglycosylation. Depending on the substrate, different quantities of reducing sugars were produced by the xylanase: 150mg/g from corncob, 105mg/g from bean culms, and 133mg/g from bagasse. With the bagasse substrate, the xylanase yielded 2.36, 2.76, 2.03, and 2.17mg/mL of X2, X3, X4, and X5, respectively. The structure of xylobiose and xylotriose from the hydrolysis of corncob xylan was identified by MS and NMR. The production of XOS from various agricultural wastes has potential industrial applications. This is the first report of XOS production by S. rameus L2001.

Thermostability improvement of Orpinomyces sp. xylanase by directed evolution

The methodology of directed evolution, using the mutagenic technique of error-prone PCR has been used to improve the thermostability of enzymes. This method was applied to the endo-β-1,4-xylanase from Orpinomyces strain PC-2. The constructed library of xylanase (xynA) mutants was subjected to several screening cycles in plates with azo-xylan-agarose as substrate and four thermostable mutants (M1–M4) were selected. Homology models for these thermostable mutants were constructed to identify the location of the residues changed by error-prone PCR and to investigate the effect of these mutations on the xylanase properties. Xylanase activities of the mutants and wild type were maximal at 60 °C and in the pH range of 5–7. The mutants displayed higher thermostability than the wild type XynA, where the wild type showed a half-life at 60 °C of 7.92 min, while half-life values for M1, M2, M3 and M4 were 209, 33.2, 401 and 15.3 min, respectively. Additionally, M3 and M4 presented a good performance in more extreme pH conditions. The mutants retained their ability to hydrolyze birchwood and oat spelt xylans, which are substrates presenting different degrees of branching.

Purification, Characterization of GH11 Endo-β-1,4-xylanase from Thermotolerant Streptomyces sp. SWU10 and Overexpression in Pichia pastoris KM71H

We have previously described two forms of an endo-β-1,4-xylanase (XynSW2A and XynSW2B) synthesized by thermotolerant Streptomyces sp. SWU10. Here, we describe another xylanolytic enzyme, designated XynSW1. The enzyme was purified to homogeneity from 2 L of culture filtrate. Its apparent molecular mass was 24 kDa. The optimal pH and temperature were pH 5.0 and 40 °C, respectively. The enzyme was stable in a wide pH ranges (pH 1-11), more than 80 % of initial activity remained at pH 2-11 after 16 h of incubation at 4 °C and stable up to 50 °C for 1 h. Xylobiose and xylotriose were the major xylooligosaccharides released from oat spelt xylan by the action of XynSW1, indicating of endo-type xylanase. The complete xynSW1 gene contains 1,011 bp in length and encode a polypeptide of 336 with 41 amino acids of signal peptide. The amino acid sequence analysis revealed that it belongs to glycoside hydrolase family 11 (GH11). The mature xynSW1 gene without signal peptide sequence was overexpressed in Pichia pastoris KM71H. The recombinant XynSW1 protein showed higher molecular mass due to the differences in glycosylation levels at the six N-glycosylation sites in the amino acid sequence and exhibited better physicochemical properties than those of the native enzyme including higher optimal temperature (60 °C), and specific activity, but lower optimal pH (4.0). Because of their stability in a wide pH ranges, both of native and recombinant enzymes of XynSW1, may have potential application in several industries including food, textile, biofuel, and also waste treatment.

Purification and characterization of an extracellular xylanase produced by the endophytic fungus, Aspergillus terreus, grown in submerged fermentation

Aspergillus terreus produced high levels of a thermotolerant extracellular xylanase and showed low cellulase activity when cultured at 30°C for 48 h, in liquid medium supplemented with wheat bran as carbon source. Xylanase was purified 45-fold to homogeneity with a recovery yield of 67% by carboxymethyl (CM)-cellulose chromatography. The enzyme, a glycoprotein with 33% of carbohydrate content, appeared as a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel with a molecular mass corresponding to 23 kDa. Optimal temperature and pH were 55°C and 4.5, respectively. The enzyme was thermotolerant at 45 and 50°C, with a half-life of 55 and 36 min, respectively. The K m was calculated as 22 mg/ml and V max as 625 mg/ml of protein using birchwood xylan as substrate. Metal ions, such as Ag + , Cu +2 , Fe +2 , Hg + and Zn +2 strongly inhibited xylanase, whereas K + and Mn +2 resulted in activation. Xylanase hydrolyzed birchwood xylan and oatspelt xylan, mostly yielding xylooligosaccharides, suggest that it is an endoxylanase (EC. 3.2.1.37). Keywords: Aspergillus terreus , endoxylanase, thermostability

Purification and biochemical characterization of two major thermophilic xylanase isoforms (T70 and T90) from xerophytic Opuntia vulgaris plant spp.

Thermostable xylanase isoforms T70 and T90 were purified and characterized from the xerophytic Opuntia vulgaris plant species. The enzyme was purified to homogeneity employing three consecutive steps. The purified T70 and T90 isoforms yielded a final specific activity 134.0 and 150.8 U mg−1 protein, respectively. The molecular mass of these isoforms was determined to be 27 kDa. The optimum pH for the T70 and T90 xylanase isoforms was 5.0 and the temperature for optimal activity was 70 and 90 °C, respectively. The Km value of T70 and T90 enzyme isoforms was 3.49, 2.1 mg ml−1, respectively when oat spelt xylan was used as a substrate. The T70 had a Vmax of 10.4 μmol min−1 mg−1, and T90 had a Vmax of 8.9 μmol min−1 mg−1, respectively. In the presence of 10 mM Co2+, and Mn2+ the activity of T70 and T90 isoforms increased, where as 90 % inhibition was noted with of the use 10 mM Hg2+, Cd2+, Cu2+, Zn2+ while partial inhibition was observed in the presence of Fe3+, Ni2+, Ca2+and Mg2+. The T70 and T90 isoforms retained nearly 50 % activity in the presence of 2.0 M urea, while use of 40 mM SDS lowered the activity nearly 38–41 %. The substrate specificity of both T70 and T90 isoforms showed maximum activity for oat spelt xylan. Western blot, immunodiffusion, and in vitro inhibition assays confirmed reactivity of the T90 isoform with polyclonal anti-T90 antibody raised in rabbit, as well as cross-reactivity of the antibody with the T70 xylanase isoform.

Activity and thermostability increase of xylanase following transplantation with modules sub-divided from hyper-thermophilic CBM9_1-2

Transplantation is useful for elucidating the functions of structural modules and for engineering enzyme properties. Unexpectedly, transplanting a hyper-thermophilic carbohydrate-binding module, CBM9_1-2, into the mesophilic Aspergillus niger GH11 xylanase (Xyn) slightly decreased the thermal inactivation half-life of Xyn. This effect was further investigated by dividing the CBM9_1-2 module into two smaller parts, C1 and C2, which were transplanted into Xyn to create the chimeras Xyn-C1 and Xyn-C2. Both chimeras exhibited higher catalytic activities on xylan than native Xyn. Xyn-C2 exhibited higher binding affinities for both oat spelt and birch wood xylans, and its thermal inactivation half-life (69.3min) was 4 or 5 times longer than that of Xyn (17.6min), Xyn-C1 (13.4min), and the original chimera containing CBM9_1-2 (13.8min). In contrast, Xyn-C1 exhibited higher binding affinity for oat spelt xylan, but not for birch wood xylan. Through this rational engineering of the fungal xylanase, the C2 sub-module was shown to have a different thermostabilizing effect than the C1 sub-module. The different functions of the smaller parts of a large module can play pivotal roles in transplantation.

Cloning and constitutive expression of His-tagged xylanase GH 11 from Penicillium occitanis Pol6 in Pichia pastoris X33: Purification and characterization

High-level constitutive expression of xylanase GH11 from Penicillium occitanis Pol6 termed PoXyn2 was achieved using the methylotrophic yeast Pichia pastoris. The PoXyn2 cDNA encoding for a mature xylanase of 320 amino acids was subcloned into the pGAPZαA vector, to construct recombinant xylanse with six histidine residues at the N-terminal and further integrated into the genome of P. pastoris X-33 under the control of the glyceraldehyde 3-phosphate dehydrogenase (GAP) constitutive promoter. Activity assay and SDS–PAGE demonstrate that the His-tagged xylanase was extracellularly expressed in P. pastoris and purified to homogeneity by a simple, one-step purification protocol using immobilized metal affinity chromatography (Ni–NTA resin). The purified PoXyn2 showed a single band on SDS–PAGE with an apparent molecular weight of 30kDa. The xylanase activity was optimal at pH 3.0 and 50°C. The specific activity measured for Oat Spelt Xylan was 8549.85Umg−1. The apparent The KM and Vmax values were 8.33±0.7mgml−1and 58.82±0.9μmolmin−1ml−1, respectively, as measured on Oat Spelt Xylan. This is the first report demonstrating the possibility of mass production of P. occitanis xylanase using P. pastoris.

English

Xylanase producing thermophilic actinomycetes strain B42 was isolated from bagasse. This strain was enriched on oat spelt xylan agar medium and screened onto xylan-congo red agar plate by the xylanolysis method. The Phylogenetic analysis using 16S rDNA sequence data showed that strain B42 had the highest homology (99.0%) with&nbsp;Laceyella sacchari&nbsp;and it was named as&nbsp;L. sacchari&nbsp;strain B42.&nbsp;&nbsp;L. sacchari&nbsp;strain B42 xylanase was purified to study its biochemical characteristics and its biobleaching efficiency. The partial purification of xylanase using acetone fractionation (at 1:3.0 ratio) gave 2.51 fold purification and the recovery of 88%. Further purification of the partially purified xylanase using DEAE-Sephadex A-50 and G-100 column chromatography gave 11.41 fold purification and 22.80% yield with the specific activity of 1750.0 U/mg. The molecular mass of the purified xylanase was ~30.0 kDa, as analyzed by SDS/PAGE and zymogram. The enzyme reactions followed Michaelis&ndash;Menten kinetics with&nbsp;Km and&nbsp;Vmax values of4.166 mM&nbsp;and&nbsp;3787.87&micro;mole/min/ml/mg,&nbsp;respectively, as obtained from a Lineweaver&ndash;Burk plot. The optimal temperature of the enzyme was 70&deg;C. The enzyme retained 72% of its activity at 70&deg;C and 48% activity at 80&deg;C after 6 h of incubation. The half life (t1/2) of purified xylanase was 6 h at 80&deg;C. The optimal pH of xylanase activity was 10.0 and enzyme appeared to be stable over a broad pH range (pH, 11.0 to 12.0) under the assay conditions.&nbsp;Approximately 68 and 64% of the original activity was retained after 5 h of incubation at pH, 10.0 and 11.0, respectively.&nbsp;The enzymatic biobleaching of kraft pulp reduced ~26% kappa number, decreased 1.68% lignin content and released 24 fold reducing sugars. The enzyme also released sufficient amount of phenolic and hydrophobic compounds. The UV absorption spectrum of the compounds released by enzymatic treatment at 280 nm indicates the presence of lignin in the released coloring matter. &nbsp; Key words:&nbsp;Kappa number, lignin content, Michaelis&ndash;Menten, lineweaver&ndash;burk plot.

Cloning, purification and characterization of an alkali-stable endoxylanase from thermophilic Geobacillus sp. 71

The gene encoding a xylanase from Geobacillus sp. 71 was isolated, cloned, and sequenced. Purification of the Geobacillus sp 7.1 xylanase, XyzGeo71, following overexpression in E. coli produced an enzyme of 47 kDa with an optimum temperature of 75°C. The optimum pH of the enzyme is 8.0, but it is active over a broad pH range. This protein showed the highest sequence identity (93%) with the xylanase from Geobacillus thermodenitrificans NG80-2. XyzGeo71 contains a catalytic domain that belongs to the glycoside hydrolase family 10 (GH10). XyzGeo71 exhibited good pH stability, remaining stable after treatment with buffers ranging from pH 7.0 to 11.0 for 6 h. Its activity was partially inhibited by Al(3+) and Cu(2+) but strongly inhibited by Hg(2+). The enzyme follows Michaelis-Menten kinetics, with K(m) and V(max) values of 0.425 mg xylan/ml and 500 μmol/min.mg, respectively. The enzyme was free from cellulase activity and degraded xylan in an endo fashion. The action of the enzyme on oat spelt xylan produced xylobiose and xylotetrose.

In situ enzymatic aided formation of xylan hydrogels and encapsulation of horse radish peroxidase for slow release

Xylans have special gelling and film forming properties for production of hydrogels that can be used as encapsulation matrices for slow release of bioactive agents. Hydrogels with particle sizes ranging from 18nm to >10μm and zeta potential of up to −19mV were formed by selective removal of arabinose side chains from oat spelt xylan by recombinant α-l-arabinofuranosidase (AbfB). The xylan concentration significantly (P<0.05) influenced both the particle size and zeta potential of the oat spelt xylan hydrogels, and the later was also influenced significantly (P<0.05) by the hydrolysis time. The horse radish peroxidase was encapsulated in situ, both during and after formation of the xylan hydrogels and was released in an active form for a period of 180min. Thus, the recombinant AbfB presents a biological tool for modification of xylan into biodegradable encapsulation matrices for slow or targeted release of bioactive agents.

Induction of xylanase in thermophilic fungi Scytalidium thermophilum and Sporotrichum thermophile

Regulation of xylanase production in two thermophilic fungi Scytalidium thermophilum and Sporotrichum thermophile was investigated. The expression of xylanase was found to be inducible in both the cases. Various carbon sources were tested so as to identify the inducers. Soy flour and oat spelt xylan induced maximum level of xylanase in Scytalidium thermophilum and Sporotrichum thermophile respectively. Induction of xylanase in Scytalidium thermophilum led to simultaneous induction of cellulase. The zymography of enzyme preparations revealed that different carbon sources caused differential expression of multiple isoforms of xylanase in Scytalidium thermophilum, but same isoforms were expressed by Sporotrichum thermophile irrespective of the carbon source used.

Bacillus subtilis SJ01 produces hemicellulose degrading multi-enzyme complexes

Cellulose and hemicellulose account for a large portion of the world’s plant biomass. In nature, these polysaccharides are intertwined, forming complex materials that require multiple enzymes to degrade them. Multi-enzyme complexes (MECs) consist of a number of enzymes working in close proximity and synergistically to degrade complex substrates with higher efficiency than individual enzymes. The aim of this study was to isolate and characterise a (hemi-) cellulolytic MEC from the aerobic bacterium, Bacillus subtilis SJ01, using ultrafiltration followed by size-exclusion chromatography on a Sephacryl S-400 column. Two MECs, C1 and C2 of 371 and 267 kDa, respectively, were purified, consisting of 16 and 18 subunits, respectively, five of which degraded birchwood and oat spelt xylan. The MECs degraded xylan substrates (C1: 0.24 U/mg, C2: 0.14 U/mg birchwood xylan) with higher efficiency than amorphous cellulose substrates (C1: 0.002 U/mg, C2: 0.01 U/mg carboxymethyl cellulose – CMC). Low or no binding to insoluble substrates indicated that the MECs lacked some of the features characteristic of cellulosomes. The significance of this study lies in the discovery of MECs that differ structurally from cellulosomes that can hydrolyse substrates with high hemicellulose content.