Oligosaccharide Intermediates Research Articles

Heterologous Expression and Characterization of a Novel Mesophilic Maltogenic α-Amylase AmyFlA from Flavobacterium sp. NAU1659.

A novel amylase AmyFlA from Flavobacterium sp. NAU1659, AmyFlA, was cloned and expressed in Esherichia coli. Based on phylogenetic and functional analysis, it was identified as a novel member of the subfamily GH13_46, sharing high sequence identity. The protein was predicted to consist of 620 amino acids, with a putative signal peptide of 25 amino acids. The enzyme was able to hydrolyze soluble starch with a specific activity of 352.97 U/mg at 50°C in 50 mM phosphate buffer (pH 6.0). The Km and Vmax values of AmyFlA were respectively 3.15mg/ml and 566.36 µmol·ml-1·min-1 under optimal conditions. Its activity towards starch was enhanced by 63% in the presence of 1 mM Ca2+, indicating that AmyFlA was a Ca2+-dependent amylase. Compared to the reported maltogenic amylases, AmyFlA produced a lower variety of intermediate oligosaccharides at the start of the reaction so that the product mixture contained a higher proportion of maltose. These results indicate that AmyFlA may be potential application value in the production of high-maltose syrup.

Substrate and Process Engineering for Biocatalytic Synthesis and Facile Purification of Human Milk Oligosaccharides.

Innovation in process development is essential for applying biocatalysis in industrial and laboratory production of organic compounds, including beneficial carbohydrates such as human milk oligosaccharides (HMOs). HMOs have attracted increasing attention for their potential application as key ingredients in products that can improve human health. To efficiently access HMOs through biocatalysis, a combined substrate and process engineering strategy is developed, namely multistep one-pot multienzyme (MSOPME) design. The strategy allows access to a pure tagged HMO in a single reactor with a single C18-cartridge purification process, despite the length of the target. Its efficiency is demonstrated in the high-yielding (71-91 %) one-pot synthesis of twenty tagged HMOs (83-155 mg), including long-chain oligosaccharides with or without fucosylation or sialylation up to nonaoses from a lactoside without the isolation of the intermediate oligosaccharides. Gram-scale synthesis of an important HMO derivative - tagged lacto-N-fucopentaose-I (LNFP-I) - proceeds in 84 % yield. Tag removal is carried out in high efficiency (94-97 %) without the need for column purification to produce the desired natural HMOs with a free reducing end. The method can be readily adapted for large-scale synthesis and automation to allow quick access to HMOs, other glycans, and glycoconjugates.

Preactivation-based chemoselective glycosylations: A powerful strategy for oligosaccharide assembly.

Most glycosylation reactions are performed by mixing the glycosyl donor and acceptor together followed by the addition of a promoter. While many oligosaccharides have been synthesized successfully using this premixed strategy, extensive protective group manipulation and aglycon adjustment often need to be performed on oligosaccharide intermediates, which lower the overall synthetic efficiency. Preactivation-based glycosylation refers to strategies where the glycosyl donor is activated by a promoter in the absence of an acceptor. The subsequent acceptor addition then leads to the formation of the glycoside product. As donor activation and glycosylation are carried out in two distinct steps, unique chemoselectivities can be obtained. Successful glycosylation can be performed independent of anomeric reactivities of the building blocks. In addition, one-pot protocols have been developed that have enabled multiple-step glycosylations in the same reaction flask without the need for intermediate purification. Complex glycans containing both 1,2-cis and 1,2-trans linkages, branched oligosaccharides, uronic acids, sialic acids, modifications such as sulfate esters and deoxy glycosides have been successfully synthesized. The preactivation-based chemoselective glycosylation is a powerful strategy for oligosaccharide assembly complementing the more traditional premixed method.

Formal Synthesis of Anticoagulant Drug Fondaparinux Sodium.

The practical formal synthesis of the anticoagulant drug fondaparinux sodium 1 was accomplished using an optimized modular synthetic strategy. The important pentasaccharide 2, a precursor for the synthesis of fondaparinux sodium, was synthesized on a 10 g scale in 14 collective steps with 3.5% overall yield from well-functionalized monosaccharide building blocks. The strategy involved a convergent [3 + 2] coupling approach, with excellent stereoselectivity in every step of glycosylation from the monosaccharide building blocks. Efficient routes to the syntheses of these fully functionalized building blocks were developed, minimizing oligosaccharide stage functional-group modifications. The syntheses of all building blocks avoided rigorous reaction conditions and the use of expensive reagents. In addition, common intermediates and a series of one-pot reactions were employed to enhance synthetic efficiency, improving the yield considerably. In the monosaccharide-to-oligosaccharide assembly reactions, cheaper activators (e.g., NIS/TfOH, TESOTf, and TfOH) were used to facilitate highly efficient glycosylations. Furthermore, crystallization of several monosaccharide and oligosaccharide intermediates significantly simplified purification procedures, which would be greatly beneficial to the scalable synthesis of fondaparinux sodium.

Cloning, expression and characterization of the recombinant cold-active type-I pullulanase from Shewanella arctica

An activity-based screening approach led to the identification of a novel glycoside hydrolase family-13 pullulanase gene (pul13A) from a psychrophilic bacterium. The recombinant enzyme exhibited a deduced peptide sequence of 1440 amino acid residues and was produced in a heterologous host in Escherichia coli. Purification from inclusion bodies was achieved by a six-step dialysis protocol enabling mild refolding of the urea-denaturated protein followed by affinity- and size-exclusion chromatography under native conditions. Pul13A is a type-I pullulanase, which is capable of hydrolysing α-1,6-glycosidic bonds in pullulan to produce maltotriose, while maltose and intermediate oligosaccharides are produced from soluble starch and amylopectin. The recombinant enzyme exhibited typical properties of cold-adapted proteins including low thermostability at elevated temperatures. It showed a temperature optimum at 35°C, while at 10°C residual activity (25%) remained. The optimal pH was in the range of 6.0–7.0, with Pul13A being stable at neutral and basic pH, but not in the acidic range. Catalytic activity was increased in the presence of divalent cations calcium and cobalt and both metal ions were also able to restore catalytic activity of EDTA-chelated enzyme samples. Pul13A represents the first type-I pullulanase from a psychrophile that has been produced in recombinant form. Moreover, its favourable enzymatic properties make this enzyme a potential candidate for industrial applications such as starch degradation for ethanol based biofuel production.

Structural basis for substrate recognition mechanism of ER glucosidase II

The endoplasmic reticulum (ER) possesses a sophisticated quality control system to proofread newly synthesized proteins. A series of N-linked oligosaccharide intermediates attached on the nascent proteins serves as specific tags for the quality control system. In this system, glucosidase II is involved in trimming of non-reducing terminal glucose residue of N-glycan intermediates. Glucosidase II consists of approximately 110 kDa catalytic α subunit (GIIα) and 60 kDa non-catalytic regulatory β subunit (GIIβ). It has been shown that GIIα alone can hydrolyze a small α-glycosidase model substrate (pNP-glucose), while it cannot catalyze deglucosylation of the N-linked oligosaccharide substrates unless it makes a complex with GIIβ. In this study, we determined the first crystal structure of GIIα in the absence and presence of its inhibitor 1-deoxynojirimycin at 1.6-Å resolution. The crystal structure revealed that GIIα has a characteristic segment at the N-terminus as compared with the cognate glycoside hydrolases (GH31). Interestingly, the N-terminal segment was accommodated on the substrate-binding pocket. Based on these results, we suggest that the N-terminal segment of GIIα undergoes structural rearrangement through interaction with GIIβ, thereby promoting the substrate-binding capacity for the N-linked oligosaccharide substrates.

Synthesis and characterization of acetylated sept-d-glucopyranose carbamate as an oligosaccharide donor

Abstract An oligosaccharide donor, acetylated sept- d -glucopyranose tetradecyl carbamate, was designed and synthesized. This compound could be easily linked to hydroxyl-containing compounds through an O -glycosidic bond. Characterization of all the oligosaccharide intermediates and the final product was thoroughly discussed.

Characterization of an α-glucosidase, HdAgl, from the digestive fluid of Haliotis discus hannai

Previously, we isolated two α-amylase isozymes, HdAmy58 and HdAmy82, from the digestive fluid of the Pacific abalone Haliotis discus hannai (Kumagai et al., 2013, Comp. Biochem. Physiol., B 164, 80-88). These enzymes degraded starch producing maltooligosaccharides but not glucose. However, the digestive fluid itself could produce glucose from starch, indicating that the digestive fluid contains α-glucosidase-like enzymes together with the α-amylases. Thus, in the present study, we isolated this α-glucosidase-like enzyme from the digestive fluid and characterized it to some extent. Isolation of this enzyme was carried out by ammonium sulfate fractionation followed by conventional column chromatographies and FPLC. The purified enzyme showed an apparent molecular mass of 97kDa on SDS-PAGE, and optimal temperature and pH of 45°C and 3.8-5.5, respectively. This enzyme could degrade various sizes of maltooligosaccharides into glucose and released glucose from starch producing no appreciable intermediate oligosaccharides. We concluded that this enzyme is an α-glucosidase (EC 3.2.1.20) exolitically acting on polymer substrate and named HdAgl. HdAgl efficiently degraded maltose but hardly degraded p-nitrophenyl α-D-glucopyranoside (α-pNPG) and isomaltose. This enzyme was regarded as a maltase-like α-glucosidase that preferably degrades maltose but scarcely aryl glucosides. When starch was used as a substrate, HdAgl converted approximately 40% (w/w) of the starch to glucose. If an abalone α-amlylase HdAmy58 was added to the reaction mixture, the glucose yield increased to 60% (w/w). These results suggested that both HdAgl and HdAmy58 play important roles for the production of glucose from dietary starch in the digestive fluid. The amino-acid sequence of 887 residues for HdAgl was deduced by the cDNA method. This sequence showed 41-46% amino-acid identities to those of mammalian and avian α-glucosidases belonging to glycoside-hydrolase-family31.

A Small Multidrug Resistance-like Transporter Involved in the Arabinosylation of Arabinogalactan and Lipoarabinomannan in Mycobacteria

The biosynthesis of the major cell envelope glycoconjugates of Mycobacterium tuberculosis is topologically split across the plasma membrane, yet nothing is known of the transporters required for the translocation of lipid-linked sugar donors and oligosaccharide intermediates from the cytoplasmic to the periplasmic side of the membrane in mycobacteria. One of the mechanisms used by prokaryotes to translocate lipid-linked phosphate sugars across the plasma membrane relies on translocases that share resemblance with small multidrug resistance transporters. The presence of an small multidrug resistance-like gene, Rv3789, located immediately upstream from dprE1/dprE2 responsible for the formation of decaprenyl-monophosphoryl-β-D-arabinose (DPA) in the genome of M. tuberculosis led us to investigate its potential involvement in the formation of the major arabinosylated glycopolymers, lipoarabinomannan (LAM) and arabinogalactan (AG). Disruption of the ortholog of Rv3789 in Mycobacterium smegmatis resulted in a reduction of the arabinose content of both AG and LAM that accompanied the accumulation of DPA in the mutant cells. Interestingly, AG and LAM synthesis was restored in the mutant not only upon expression of Rv3789 but also upon that of the undecaprenyl phosphate aminoarabinose flippase arnE/F genes from Escherichia coli. A bacterial two-hybrid system further indicated that Rv3789 interacts in vivo with the galactosyltransferase that initiates the elongation of the galactan domain of AG. Biochemical and genetic evidence is thus consistent with Rv3789 belonging to an AG biosynthetic complex, where its role is to reorient DPA to the periplasm, allowing this arabinose donor to then be used in the buildup of the arabinan domains of AG and LAM.

Challenging Deprotection Steps During the Synthesis of Tetra- and Pentasaccharide Fragments of the LeaLex Tumor-Associated Hexasaccharide Antigen

We report the convergent synthesis of two novel tetrasaccharide and two novel pentasaccharide fragments of the Le(a)Le(x) TACA: the tetrasaccharides contain neither the galactose at the Le(a) nonreducing end nor the fucose at the Le(x) reducing end; the pentasaccharides only lack the galactose residue at the Le(a) nonreducing end. Two of the analogues were prepared as hexyl glycosides to be used in NMR experiments and as soluble inhibitors in binding studies and two as 6-aminohexyl glycosides to be conjugated to carrier proteins. Our strategy relied on stepwise extensions using excess monosaccharide glycosyl donors (trichloroacetimidates and thioglycosides) in sequential glycosylation reactions. The protecting groups were chosen to limit the number of deprotection steps required to obtain the final derivatives. While this strategy ensured that all glycosylation reactions proceeded in very good yields (70-84%), deprotection of the oligosaccharide intermediates was challenging. Global deprotection using Birch metal dissolving conditions did not remove the tert-butyldiphenylsilyl group, which indeed was incompatible with such reaction conditions. Attempts to remove the TBDPS with tetrabutylammonium fluoride was unsuccessful and led to a complex mixture of compounds that could not be separated. The desired hexyl and aminohexyl tetrasaccharides were finally obtained after four- and five-step deprotection sequences, respectively. Deprotection of the pentasaccharide intermediate to give the hexyl and aminohexyl analogues also led to unexpected results. Indeed, during Zemplén deacylation, a chloroacetamide chlorine atom was displaced by methoxide ions leading to the corresponding methoxyacetamide. Once the chloroacetamide was fully reduced to an acetamide the pentasaccharides were obtained in four and five steps, respectively.

Microwave-assisted sulfonation of heparin oligosaccharides

The use of microwaves for the efficient and fast O-sulfonation of heparin oligosaccharide intermediates has been reported for the first time. Experimental problems typically associated with this chemical reaction, such as poor isolated yields and long reaction times, have been avoided with the present method. The efficiency of this protocol was demonstrated by the high-yielding sulfonation of a series of oligosaccharides using SO3·Me3N complex. Microwave-assisted sulfonation is expected to greatly facilitate the preparation of heparin oligosaccharides, a crucial step for understanding the role of these complex carbohydrates in biological processes.

Efficient alternative for the reduction of N-trichloroacetyl groups in synthetic chondroitin oligosaccharide intermediates

Abstract An efficient alternative for the reduction of N -trichloroacetyl groups in chondroitin oligosaccharides is reported. It involves the use of Zn–Cu couple in acetic acid, and was successfully applied to a large panel of synthetic intermediates useful for the preparation of chondroitin oligomers, a class of natural products with numerous relevant biological functions.

Analysis of Bacterial Lipid-Linked Oligosaccharide Intermediates Using Porous Graphitic Carbon Liquid Chromatography-Electrospray Ionization Mass Spectrometry: Heterogeneity in the Polyisoprenyl Carrier Revealed

N-glycosylation of proteins is recognized as one of the most common post-translational modifications. It was believed that N-glycosylation occurred exclusively in eukaryotes until the recent discovery of the general protein glycosylation pathway (Pgl) in Campylobacter jejuni, which has similarities to the eukaryotic system and adds proteins en bloc from a lipid carrier to a protein acceptor. In addition to N-linked glycans, a number of pathogenic bacteria such as Pseudomonas aeruginosa and Neisseria species have been shown to O-glycosylate their proteins through polyisoprene-linked intermediates. To date, most techniques to analyze lipid-linked oligosaccharides (LLOs) of these pathways involve the use of radiolabels and chromatographic separation. With the increasing frequency of reports of bacterial protein glycosylation that proceed through lipid-mediated steps, there is a need for technologies capable of characterizing these newly described bacterial systems as well as eukaryotic pathways from biologically relevant samples in an accurate, rapid, and cost-effective manner. In this paper, a new glycomics strategy based on porous graphite carbon (PGC) liquid chromatography mass spectrometry (LC-MS) was devised and validated on the C. jejuni N-glycan pathway. Lipid-linked oligosaccharide intermediates of the Pgl pathway from crude lipid extracts were separated using online chromatography on a capillary PGC column with a chloroform gradient. By exploiting the retention properties of hydrophobic and polar analytes on PGC, baseline separation of LLOs with minor changes in oligosaccharide structure and polyisoprene chain length was obtained. This method is capable of analyzing low levels of LLOs (from approximately 10(6) bacterial cells) and distinguishing the LLOs that differ by as little as one monosaccharide or polyisoprene unit. Furthermore, we have demonstrated for the first time that oligosaccharides of the C. jejuni Pgl pathway are assembled on different polyisoprenes, e. g. C(45), C(60), and apparent hydroxylated forms, in addition to those previously reported (i.e., C(50) and C(55)). The hydroxylated forms of the LLOs are believed to be an intermediate in the degradation of accumulated LLOs for polyisoprene carrier recycling.

Pre-activation-based one-pot synthesis of an alpha-(2,3)-sialylated core-fucosylated complex type bi-antennary N-glycan dodecasaccharide.

Synthesis of N-glycans is of high current interests due to their important biological properties. A highly efficient convergent strategy based on the pre-activation method for assembly of the complex type core fucosylated bi-antennary N-glycan dodecasaccharide has been developed. Retrosynthetically, this extremely challenging target is broken down to three modules: a sialyl disaccharide, a glucosamine building block and a hexasaccharide diol acceptor. The sialyl disaccharide was easily obtained by selective activation of a new 5-N-trichloroacetyl protected sialyl donor in the presence of a thiogalactoside acceptor. The hexasaccharide diol module was produced by double mannosylation of a fucosylated tetrasaccharide acceptor, which in turn was generated by glycosylation of a alpha-fucosylated disaccharide with a beta-mannose containing disaccharide donor. The union of the three modules was performed in one-pot giving the fully protected dodecasaccharide in high yield. This synthesis is characterized by minimum protective group and aglycon adjustment on oligosaccharide intermediates, thus greatly enhancing the overall synthetic efficiency. The modular feature of this strategy suggests that this method can be readily adapted to the synthesis of a wide variety of N-glycan structures.

Affinity-Capture Tandem Mass Spectrometric Characterization of Polyprenyl-Linked Oligosaccharides: Tool to Study Protein N-Glycosylation Pathways

N-glycosylation of proteins is recognized as one of the most common post-translational modifications. Until recently it was believed that N-glycosylation occurred exclusively in eukaryotes before the discovery of the general protein glycosylation pathway (Pgl) in Campylobacter jejuni. To date, most techniques to analyze lipid-linked oligosaccharides (LLOs) of these pathways involve the use of radiolabels and chromatographic separation. Technologies capable of characterizing eukaryotic and the newly described bacterial N-glycosylation systems from biologically relevant samples in a quick, accurate, and cost-effective manner are needed. In this paper a new glycomics strategy based on lectin-affinity capture was devised and validated on the C. jejuni N-glycan pathway and the engineered Escherichia coli strains expressing the functional C. jejuni pathway. The lipid-linked oligosaccharide intermediates of the Pgl pathway were then enriched using SBA-agarose affinity-capture and examined by capillary electrophoresis-mass spectrometry (CE-MS). We demonstrate that this method is capable of detecting low levels of LLOs, the sugars are indeed assembled on undecaprenylpyrophosphate, and structural information for expected and unexpected LLOs can be obtained without further sample manipulation. Furthermore, CE-MS analyses of C. jejuni and the E. coli "glyco-factories" showed striking differences in the assembly and control of N-glycan biosynthesis.

Highly efficient syntheses of hyaluronic acid oligosaccharides.

Highly efficient syntheses of hyaluronic acid oligosaccharides have been accomplished through the pre-activation based iterative one-pot strategy. A series of oligosaccharides ranging from di- to hexasaccharides were rapidly assembled using only near stoichiometric amounts of the building blocks without aglycon adjustment or purifications of intermediate oligosaccharides. Deprotection and oxidation protocols were developed for protective group removal and oxidation-state adjustment. The availability of such structurally well defined synthetic hyaluronic acid oligosaccharides will greatly facilitate the establishment of detailed structure-function relationships.

A novel oligoalginate lyase from abalone, Haliotis discus hannai, that releases disaccharide from alginate polymer in an exolytic manner

We previously reported the isolation and cDNA cloning of an endolytic alginate lyase, HdAly, from abalone Haliotis discus hannai [ Carbohydr. Res. 2003, 338, 2841–2852]. Although HdAly preferentially degraded mannuronate-rich substrates, it was incapable of degrading unsaturated oligomannuronates smaller than tetrasaccharide. In the present study, we used conventional chromatographic techniques to isolate a novel unsaturated-trisaccharide-degrading enzyme, named HdAlex, from the digestive fluid of the abalone. The HdAlex showed a molecular weight of 32,000 on SDS-PAGE and could degrade not only unsaturated trisaccharide but also alginate and mannuronate-rich polymers at an optimal pH and temperature of 7.1 and 42 °C, respectively. Upon digestion of alginate polymer, HdAlex decreased the viscosity of the alginate at a slower rate than did HdAly, producing only unsaturated disaccharide without any intermediate oligosaccharides. These results indicate that HdAlex degrades the alginate polymer in an exolytic manner. Because HdAlex split saturated trisaccharide producing unsaturated disaccharide, we considered that this enzyme cleaved the alginate at the second glycoside linkage from the reducing terminus. The primary structure of HdAlex was deduced with cDNAs amplified from an abalone hepatopancreas cDNA library by the polymerase chain reaction. The translational region of 822 bp in the total 887-bp sequence of HdAlex cDNA encoded an amino-acid sequence of 273 residues. The N-terminal sequence of 16 residues, excluding the initiation methionine, was regarded as the signal peptide of this enzyme. The amino-acid sequence of the remaining 256 residues shared 62–67% identities with those of the polysaccharide lyase family-14 (PL14) enzymes such as HdAly and turban-shell alginate lyase SP2. To our knowledge, HdAlex is the first exolytic oligoalginate lyase belonging to PL14.

Two distinct but interchangeable mechanisms for flipping of lipid-linked oligosaccharides

Translocation of lipid-linked oligosaccharide (LLO) intermediates across membranes is an essential but poorly understood process in eukaryotic and bacterial glycosylation pathways. Membrane proteins defined as translocases or flippases are implicated to mediate the translocation reaction. The membrane protein Wzx has been proposed to mediate the translocation across the plasma membrane of lipopolysaccharide (LPS) O antigen subunits, which are assembled on an undecaprenyl pyrophosphate lipid carrier. Similarly, PglK (formerly WlaB) is a Campylobacter jejuni-encoded ABC-type transporter proposed to mediate the translocation of the undecaprenylpyrophosphate-linked heptasaccharide intermediate involved in the recently identified bacterial N-linked protein glycosylation pathway. A combination of genetic and carbohydrate structural analyses defined and characterized flippase activities in the C. jejuni N-linked protein glycosylation and the Escherichia coli LPS O antigen biosynthesis. PglK displayed relaxed substrate specificity with respect to the oligosaccharide structure of the LLO intermediate and complemented a wzx deficiency in E. coli O-antigen biosynthesis. Our experiments provide strong genetic evidence that LLO translocation across membranes can be catalyzed by two distinct proteins that do not share any sequence similarity.

Substrate Specificity and Domain Functions of Extracellular Heparan Sulfate 6-O-Endosulfatases, QSulf1 and QSulf2

The extracellular sulfatases (Sulfs) are an evolutionally conserved family of heparan sulfate (HS)-specific 6-O-endosulfatases. These enzymes remodel the 6-O-sulfation of cell surface HS chains to promote Wnt signaling and inhibit growth factor signaling for embryonic tissue patterning and control of tumor growth. In this study we demonstrate that the avian HS endosulfatases, QSulf1 and QSulf2, exhibit the same substrate specificity toward a subset of trisulfated disaccharides internal to HS chains. Further, we show that both QSulfs associate exclusively with cell membrane and are enzymatically active on the cell surface to desulfate both cell surface and cell matrix HS. Mutagenesis studies reveal that conserved amino acid regions in the hydrophilic domains of QSulf1 and QSulf2 have multiple functions, to anchor Sulf to the cell surface, bind to HS substrates, and to mediate HS 6-O-endosulfatase enzymatic activity. Results of our current studies establish the hydrophilic domain (HD) of Sulf enzymes as an essential multifunctional domain for their unique endosulfatase activities and also demonstrate the extracellular activity of Sulfs for desulfation of cell surface and cell matrix HS in the control of extracellular signaling for embryonic development and tumor progression.

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 β-(1 → 3)/β-(1 → 4) linkages, were analysed by physicochemical and biochemical techniques. With the glycoside hydrolase family 10 β-(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 β-(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 β-(1 → 3)/β-(1 → 4) linkage distribution within the linear d-xylose polymer. Similar results were obtained with rhodymenan, the seaweed xylan from Palmares palmata.