Substrate specificity mapping of fungal CAZy AA3_2 oxidoreductases.

BACKGROUND: Oxidative enzymes targeting lignocellulosic substrates are presently classified into various auxiliary activity (AA) families within the carbohydrate-active enzyme (CAZy) database. Among these, the fungal AA3 glucose-methanol-choline (GMC) oxidoreductases with varying auxiliary activities are attractive sustainable biocatalysts and important for biological function. CAZy AA3 enzymes are further subdivided into four subfamilies, with the large AA3_2 subfamily displaying diverse substrate specificities. However, limited numbers of enzymes in the AA3_2 subfamily are currently biochemically characterized, which limits the homology-based mining of new AA3_2 oxidoreductases. Importantly, novel enzyme activities may be discovered from the uncharacterized parts of this large subfamily. RESULTS: In this study, phylogenetic analyses employing a sequence similarity network (SSN) and maximum likelihood trees were used to cluster AA3_2 sequences. A total of 27 AA3_2 proteins representing different clusters were selected for recombinant production. Among them, seven new AA3_2 oxidoreductases were successfully produced, purified, and characterized. These enzymes included two glucose dehydrogenases (TaGdhA and McGdhA), one glucose oxidase (ApGoxA), one aryl alcohol oxidase (PsAaoA), two aryl alcohol dehydrogenases (AsAadhA and AsAadhB), and one novel oligosaccharide (gentiobiose) dehydrogenase (KiOdhA). Notably, two dehydrogenases (TaGdhA and KiOdhA) were found with the ability to utilize phenoxy radicals as an electron acceptor. Interestingly, phenoxy radicals were found to compete with molecular oxygen in aerobic environments when serving as an electron acceptor for two oxidases (ApGoxA and PsAaoA), which sheds light on their versatility. Furthermore, the molecular determinants governing their diverse enzymatic functions were discussed based on the homology model generated by AlphaFold. CONCLUSIONS: The phylogenetic analyses and biochemical characterization of AA3_2s provide valuable guidance for future investigation of AA3_2 sequences and proteins. A clear correlation between enzymatic function and SSN clustering was observed. The discovery and biochemical characterization of these new AA3_2 oxidoreductases brings exciting prospects for biotechnological applications and broadens our understanding of their biological functions.

Characterization of a novel AA3_1 xylooligosaccharide dehydrogenase from Thermothelomyces myriococcoides CBS 398.93.

BACKGROUND: The Carbohydrate-Active enZymes (CAZy) auxiliary activity family 3 (AA3) comprises flavin adenine dinucleotide-dependent (FAD) oxidoreductases from the glucose-methanol-choline (GMC) family, which play auxiliary roles in lignocellulose conversion. The AA3 subfamily 1 predominantly consists of cellobiose dehydrogenases (CDHs) that typically comprise a dehydrogenase domain, a cytochrome domain, and a carbohydrate-binding module from family 1 (CBM1). RESULTS: In this work, an AA3_1 gene from T. myriococcoides CBS 398.93 encoding only a GMC dehydrogenase domain was expressed in Aspergillus niger. Like previously characterized CDHs, this enzyme (TmXdhA) predominantly accepts linear saccharides with ß-(1 → 4) linkage and targets the hydroxyl on the reducing anomeric carbon. TmXdhA was distinguished, however, by its preferential activity towards xylooligosaccharides over cellooligosaccharides. Amino acid sequence analysis showed that TmXdhA possesses a glutamine at the substrate-binding site rather than a threonine or serine that occupies this position in previously characterized CDHs, and structural models suggest the glutamine in TmXdhA could facilitate binding to pentose sugars. CONCLUSIONS: The biochemical analysis of TmXdhA revealed a catalytic preference for xylooligosaccharide substrates. The modeled structure of TmXdhA provides a reference for the screening of oxidoreductases targeting xylooligosaccharides. We anticipate TmXdhA to be a good candidate for the conversion of xylooligosaccharides to added-value chemicals by its exceptional catalytic ability.

Carbohydrate esterase family 16 contains fungal hemicellulose acetyl esterases (HAEs) with varying specificity.

Acetyl esterases are an important component of the enzymatic machinery fungi use to degrade plant biomass and are classified in several Carbohydrate Esterase families of the CAZy classification system. Carbohydrate Esterase family 16 (CE16) is one of the more recently discovered CAZy families, but only a small number of its enzyme members have been characterized so far, revealing activity on xylan-derived oligosaccharides, as well as activity related to galactoglucomannan. The number of CE16 genes differs significantly in the genomes of filamentous fungi. In this study, four CE16 members were identified in the genome of Aspergillus niger NRRL3 and it was shown that they belong to three of the four phylogenetic Clades of CE16. Significant differences in expression profiles of the genes and substrate specificity of the enzymes were revealed, demonstrating the diversity within this family of enzymes. Detailed characterization of one of these four A. niger enzymes (HaeA) demonstrated activity on oligosaccharides obtained from acetylated glucuronoxylan, galactoglucomannan and xyloglucan, thus establishing this enzyme as a general hemicellulose acetyl esterase. Their broad substrate specificity makes these enzymes highly interesting for biotechnological applications in which deacetylation of polysaccharides is required.

Separation of isomeric cereal-derived arabinoxylan-oligosaccharides by collision induced dissociation-travelling wave ion mobility spectrometry-tandem mass spectrometry (CID-TWIMS-MS/MS).

The potential of travelling wave ion mobility spectroscopy in combination with collision induced dissociation tandem mass spectrometry (CID-TWIMS-MS/MS) to separate cereal-derived isomeric arabinoxylan-oligosaccharides (A)XOS was investigated. Three trisaccharide, four tetrasaccharide, and four pentasaccharide (A)XOS isomers were analyzed by positive and negative ionization TWIMS-MS and CID-TWIMS-MS/MS. The tri- and pentasaccharide isomers were distinguishable by the ATDs of the precursor ions. The CID-TWIMS-MS/MS could separate most of the isomeric fragment ions produced from tetra- and pentasaccharide (A)XOS. Finally, the base peak mobility spectrum is introduced as a practical tool for (A)XOS fingerprinting.

Valorization of Urban Street Tree Pruning Residues in Biorefineries by Steam Refining: Conversion Into Fibers, Emulsifiers, and Biogas.

Street tree pruning residues are a widely available and currently undervalorized bioresource. Their utilization could help alleviate an increasing biomass shortage and offset costs of the pruning process for the municipalities. In this work, a holistic valorization pathway of pruning residues leading to fibers, oligosaccharides, biogas, and compost is presented. For this, representative mixtures of tree pruning materials from the most prevalent street tree genera (oak, linden, maple) found in Hamburg (Germany) were prepared by shredding and cleaning procedures. Collection of sample material was performed in summer and winter to account for seasonality. A steam-based fractionation was conducted using treatment severities ranging from log R0 = 2.5 to 4.0. At the highest severity, a fiber yield of around 66%, and liquor yield of 26-30% was determined. The fibers were evaluated with respect to their properties for paper product applications, with higher treatment severities leading to higher paper strengths. From the oligosaccharide-rich liquor, emulsions were created, which showed promising stability properties over 8 weeks of storage. The liquors and the rejects from the material preparation also displayed good potential for biomethane production. Overall, the differences between material collected in summer and winter were found to be small, indicating the possibility for a year-round utilization of pruning residues. For the presented utilization pathway, high severity treatments were the most promising, featuring a high liquor yield, good biomethane potential, and the highest paper strengths.

Structural characterization of the family GH115 α-glucuronidase from Amphibacillus xylanus yields insight into its coordinated action with α-arabinofuranosidases.

The coordinated action of carbohydrate-active enzymes has mainly been evaluated for the purpose of complete saccharification of plant biomass (lignocellulose) to sugars. By contrast, the coordinated action of accessory hemicellulases on xylan debranching and recovery is less well characterized. Here, the activity of two family GH115 α-glucuronidases (SdeAgu115A from Saccharophagus degradans, and AxyAgu115A from Amphibacillus xylanus) on spruce arabinoglucuronoxylan (AGX) was evaluated in combination with an α-arabinofuranosidase from families GH51 (AniAbf51A, aka E-AFASE from Aspergillus niger) and GH62 (SthAbf62A from Streptomyces thermoviolaceus). The α-arabinofuranosidases boosted (methyl)-glucuronic acid release by SdeAgu115A by approximately 50 % and 30 %, respectively. The impact of the α-arabinofuranosidases on AxyAgu115A activity was comparatively low, motivating its structural characterization. The crystal structure of AxyAgu115A revealed increased length and flexibility of the active site loop compared to SdeAgu115A. This structural difference could explain the ability of AxyAgu115A to accommodate more highly substituted arabinoglucuronoxylan, and inform enzyme selections for improved AGX recovery and use.

Colloidal features of softwood galactoglucomannans-rich extract.

Development of a sustainable bioeconomy requires valorization of renewable resources, such as wood hemicelluloses. The intra- and inter-molecular association of hemicelluloses within themselves or with other wood components can result in complex macromolecular features. These features exhibit functionality as hydrocolloids, however macromolecular characterization of these heterogeneous materials are challenging using conventional techniques such as size-exclusion chromatography. We studied galactoglucomannans (GGM) -rich softwood extracts at two grades of purity-as crude extract and after ethanol-precipitation. Asymmetrical flow field-flow fractionation (AF4) was optimized and utilized to fractionate size classes in GGM extracts, and subsequent characterization was performed with light scattering and microscopy techniques. Both GGM extracts contained polysaccharides of around 10,000 g/mol molar mass, and colloidal assemblies and/or particles in sub-micron size range. The optimized AF4 method facilitates the characterization of complex biomass-derived carbohydrates without pre-fractionation, and provides valuable understanding of their unique macromolecular features for their future application in food, pharmaceuticals, and cosmetics.

Hybrid Aspen Expressing a Carbohydrate Esterase Family 5 Acetyl Xylan Esterase Under Control of a Wood-Specific Promoter Shows Improved Saccharification.

Fast-growing broad-leaf tree species can serve as feedstocks for production of bio-based chemicals and fuels through biochemical conversion of wood to monosaccharides. This conversion is hampered by the xylan acetylation pattern. To reduce xylan acetylation in the wood, the Hypocrea jecorina acetyl xylan esterase (HjAXE) from carbohydrate esterase (CE) family 5 was expressed in hybrid aspen under the control of the wood-specific PtGT43B promoter and targeted to the secretory pathway. The enzyme was predicted to deacetylate polymeric xylan in the vicinity of cellulose due to the presence of a cellulose-binding module. Cell-wall-bound protein fractions from developing wood of transgenic plants were capable of releasing acetyl from finely ground wood powder, indicative of active AXE present in cell walls of these plants, whereas no such activity was detected in wild-type plants. The transgenic lines grew in height and diameter as well as wild-type trees, whereas their internodes were slightly shorter, indicating higher leaf production. The average acetyl content in the wood of these lines was reduced by 13%, mainly due to reductions in di-acetylated xylose units, and in C-2 and C-3 mono-acetylated xylose units. Analysis of soluble cell wall polysaccharides revealed a 4% reduction in the fraction of xylose units and an 18% increase in the fraction of glucose units, whereas the contents of cellulose and lignin were not affected. Enzymatic saccharification of wood from transgenic plants resulted in 27% higher glucose yield than for wild-type plants. Brunauer-Emmett-Teller (BET) analysis and Simons' staining pointed toward larger surface area and improved cellulose accessibility for wood from transgenic plants compared to wood from wild-type plants, which could be achieved by HjAXE deacetylating xylan bound to cellulose. The results show that CE5 family can serve as a source of enzymes for in planta reduction of recalcitrance to saccharification.

Quantitative Comparison of Pyranose Dehydrogenase Action on Diverse Xylooligosaccharides.

Pyranose dehydrogenases (PDHs; EC 1.1.99.29; AA3_2) demonstrate ability to oxidize diverse carbohydrates. Previous studies of these enzymes have also uncovered substrate-dependent regioselectivity, along with potential to introduce more than one carbonyl into carbohydrate substrates. Enzymatic oxidation of carbohydrates facilitates their further derivatization or polymerization into bio-based chemicals and materials with higher value; accordingly, PDHs that show activity on xylooligosaccharides could offer a viable approach to extract higher value from hemicelluloses that are typically fragmented during biomass processing. In this study, AbPDH1 from Agaricus bisporus and AmPDH1 from Leucoagaricus meleagris were tested using linear xylooligosaccharides, along with xylooligosaccharides substituted with either arabinofuranosyl or 4-O-(methyl)glucopyranosyluronic acid residues with degree of polymerization of two to five. Reaction products were characterized by HPAEC-PAD to follow substrate depletion, UPLC-MS-ELSD to quantify the multiple oxidation products, and ESI-MSn to reveal oxidized positions. A versatile method based on product reduction using sodium borodeuteride, and applicable to carbohydrate oxidoreductases in general, was established to facilitate the identification and quantification of oxidized products. AbPDH1 activity toward the tested xylooligosaccharides was generally higher than that measured for AmPDH1. In both cases, activity values decreased with increasing length of the xylooligosaccharide and when using acidic rather than neutral substrates; however, AbPDH1 fully oxidized all linear xylooligosaccharides, and 60-100% of all substituted xylooligosaccharides, after 24 h under the tested reaction conditions. Oxidation of linear xylooligosaccharides mostly led to double oxidized products, whereas single oxidized products dominated in reactions containing substituted xylooligosaccharides. Notably, oxidation of specific secondary hydroxyls vs. the reducing end C-1 depended on both the enzyme and the substrate. For all substrates, however, oxidation by both AbPDH1 and AmPDH1 was clearly restricted to the reducing and non-reducing xylopyranosyl residues, where increasing the length of the xylooligosaccharide did not lead to detectable oxidation of internal xylopyranosyl substituents. This detailed analysis of AbPDH1 and AmPDH1 action on diverse xylooligosaccharides reveals an opportunity to synthesize bifunctional molecules directly from hemicellulose fragments, and to enrich for specific products through appropriate PDH selection.

Active food packaging through controlled in situ production and release of hexanal.

Transportation and storage of vegetables and fruits, including berries, is increasing to meet growing consumer demand for fresh foods. Ripening and softening of plant tissues may be slowed down by hexanal, a safe volatile compound that also has antimicrobial properties. Thus hexanal could be applied during the food distribution chain to slow down the spoilage of plant-based products and reduce food waste. Nonetheless, due to the rapid evaporation of hexanal, a constant supply is needed. Our aim was to develop a concept to incorporate food-grade sunflower oil in a polysaccharide aerogel matrix for controlled in situ production and release of hexanal. We compared enzyme- and light-catalyzed lipid oxidation reactions, determined the release of hexanal at different conditions, and performed storage stability tests of blueberries and cherry tomatoes. The lipid-loaded aerogels assessed here are a potential novel delivery matrix for controlled hexanal formation to extend the shelf life of plant-based products.

A family AA5_2 carbohydrate oxidase from Penicillium rubens displays functional overlap across the AA5 family.

Copper radical alcohol oxidases belonging to auxiliary activity family 5, subfamily 2 (AA5_2) catalyze the oxidation of galactose and galactosides, as well as aliphatic alcohols. Despite their broad applied potential, so far very few AA5_2 members have been biochemically characterized. We report the recombinant production and biochemical characterization of an AA5_2 oxidase from Penicillium rubens Wisconsin 54-1255 (PruAA5_2A), which groups within an unmapped clade phylogenetically distant from those comprising AA5_2 members characterized to date. PruAA5_2 preferentially oxidized raffinose over galactose; however, its catalytic efficiency was 6.5 times higher on glycolaldehyde dimer compared to raffinose. Deep sequence analysis of characterized AA5_2 members highlighted amino acid pairs correlated to substrate range and conserved within the family. Moreover, PruAA5_2 activity spans substrate preferences previously reported for AA5 subfamily 1 and 2 members, identifying possible functional overlap across the AA5 family.

Identification and structural analysis of cereal arabinoxylan-derived oligosaccharides by negative ionization HILIC-MS/MS.

Recent works provide evidence of the prebiotic potential of arabinoxylan-derived oligosaccharides (A)XOS. In this study, we developed a structural analysis for cereal-derived (A)XOS by negative ionization HILIC-MS/MS. Initially, we assessed twelve (A)XOS samples of known structures with different linkage positions and branching points by direct-infusion negative ESI-MSn. We subsequently developed the negative ion HILIC-MS/MS with a post-column addition of ammonium chloride. The selected (A)XOS represented both linear (arabinofuranosyl residue linked to the non-reducing end of xylooligosaccharide) and branched structures. Each (A)XOS sample produced a specific spectrum in negative ion ESI-MSn. By analyzing cross-ring fragment ions, we determined the linkage positions of linear (A)XOS. The presence or absence of diagnostic ions in the MS3 allowed us to detect different branches (O-2- or/and O-3-linked arabinofuranosyl with/or without O-4-linked xylopyranosyl at the non-reducing end). Furthermore, we could identify all analyzed samples by HILIC-MS/MS, based on the formed spectral library and chromatographic retention times.

Tissue-specific study across the stem reveals the chemistry and transcriptome dynamics of birch bark.

Tree bark is a highly specialized array of tissues that plays important roles in plant protection and development. Bark tissues develop from two lateral meristems; the phellogen (cork cambium) produces the outermost stem-environment barrier called the periderm, while the vascular cambium contributes with phloem tissues. Although bark is diverse in terms of tissues, functions and species, it remains understudied at higher resolution. We dissected the stem of silver birch (Betula pendula) into eight major tissue types, and characterized these by a combined transcriptomics and metabolomics approach. We further analyzed the varying bark types within the Betulaceae family. The two meristems had a distinct contribution to the stem transcriptomic landscape. Furthermore, inter- and intraspecies analyses illustrated the unique molecular profile of the phellem. We identified multiple tissue-specific metabolic pathways, such as the mevalonate/betulin biosynthesis pathway, that displayed differential evolution within the Betulaceae. A detailed analysis of suberin and betulin biosynthesis pathways identified a set of underlying regulators and highlighted the important role of local, small-scale gene duplication events in the evolution of metabolic pathways. This work reveals the transcriptome and metabolic diversity among bark tissues and provides insights to its development and evolution, as well as its biotechnological applications.

Impact of in situ produced exopolysaccharides on rheology and texture of fava bean protein concentrate.

The aim of this study was to investigate the impact of in situ produced exopolysaccharides (EPS) on the rheological and textural properties of fava bean protein concentrate (FPC). EPS (dextrans) were produced from sucrose by two lactic acid bacteria (LAB). The acidification, rheology, and texture of FPC pastes fermented with Leuconostoc pseudomesenteroides DSM 20193 and Weissella confusa VTT E-143403 (E3403) were compared. A clear improvement in rheological and textural parameters was observed in sucrose-added pastes after fermentation, especially with W. confusa VTT E3403. Only moderate proteolysis of fava bean protein during fermentation was observed. The microstructure of the protein in FPC pastes, as observed by confocal laser scanning microscopy, revealed a less continuous and denser structure in EPS-abundant pastes. The beneficial structure formed during EPS-producing fermentation could not be mimicked by simply mixing FPC, isolated dextran, lactic acid, and acetic acid with water. These results emphasize the benefits of in situ produced EPS in connection with the LAB fermentation of legume protein-rich foods. Fermentation with EPS-producing LAB is a cost-effective and clean-labeled technology to obtain tailored textures, and it can further enhance the usability of legumes in novel foods.

Enzymatic analysis of levan produced by lactic acid bacteria in fermented doughs.

Levans and inulins are fructans with mainly ß-(2→6) and ß-(2→1) linkages, respectively. Levans are produced by many lactic acid bacteria, e.g. during sourdough fermentation. Levans have shown prebiotic properties and may also function as in situ-produced hydrocolloids. So far, levan contents have been measured by acid hydrolysis, which cannot distinguish levans from e.g. inulins. In order to develop a specific analysis for levan in food matrices, a Paenibacillus amylolyticus endolevanase was combined with exoinulinase for levan hydrolysis. A separate endoinulinase treatment was used to detect the possible presence of inulin. Interfering sugars were removed by a pre-wash with aqueous ethanol. Levan content was estimated from fructose and glucose released in the hydrolysis, with a correction made for the residual fructose and glucose-containing sugars. The method was validated using wheat model doughs spiked with commercial Erwinia levan, and tested by analyzing levan content in Leuconostoc mesenteroides DSM 20343-fermented fava bean doughs.

Interactions between fava bean protein and dextrans produced by Leuconostoc pseudomesenteroides DSM 20193 and Weissella cibaria Sj 1b.

The aim of this study was to study the interactions between dextran and fava bean protein. Two dextrans produced by Leuconostoc pseudomesenteroides DSM 20193 and Weissella cibaria Sj 1b were purified and mixed with fava bean protein isolate (FPI) in water or in different buffers. The two isolated dextrans presented a typical dextran structure, mainly α-(1 → 6) linkages (above 95%) and few α-(1 → 3) branches, but they differed in molar mass and conformation. Dry-heating incubation of FPI and dextran mixture facilitated the conjugation of dextran to FPI through the Maillard reaction. Both mixed and conjugated systems were further heat-treated, and different influences of the formed covalent bonds on rheological properties were observed. The W. cibaria Sj 1b dextran had a much higher gel-strengthening ability than the Ln. pseudomesenteroides DSM 20193 dextran. The intermolecular FPI-dextran interactions played an important role in stabilizing the mixed systems at different pH.

A novel acetyl xylan esterase enabling complete deacetylation of substituted xylans.

BACKGROUND: Acetylated 4-O-(methyl)glucuronoxylan (GX) is the main hemicellulose in deciduous hardwood, and comprises a ß-(1→4)-linked xylopyranosyl (Xylp) backbone substituted by both acetyl groups and α-(1→2)-linked 4-O-methylglucopyranosyluronic acid (MeGlcpA). Whereas enzymes that target singly acetylated Xylp or doubly 2,3-O-acetyl-Xylp have been well characterized, those targeting (2-O-MeGlcpA)3-O-acetyl-Xylp structures in glucuronoxylan have remained elusive. RESULTS: An unclassified carbohydrate esterase (FjoAcXE) was identified as a protein of unknown function from a polysaccharide utilization locus (PUL) otherwise comprising carbohydrate-active enzyme families known to target xylan. FjoAcXE was shown to efficiently release acetyl groups from internal (2-O-MeGlcpA)3-O-acetyl-Xylp structures, an activity that has been sought after but lacking in known carbohydrate esterases. FjoAcXE action boosted the activity of α-glucuronidases from families GH67 and GH115 by five and nine times, respectively. Moreover, FjoAcXE activity was not only restricted to GX, but also deacetylated (3-O-Araf)2-O-acetyl-Xylp of feruloylated xylooligomers, confirming the broad substrate range of this new carbohydrate esterase. CONCLUSION: This study reports the discovery and characterization of the novel carbohydrate esterase, FjoAcXE. In addition to cleaving singly acetylated Xylp, and doubly 2,3-O-acetyl-Xylp, FjoAcXE efficiently cleaves internal 3-O-acetyl-Xylp linkages in (2-O-MeGlcpA)3-O-acetyl-Xylp residues along with densely substituted and branched xylooligomers; activities that until now were missing from the arsenal of enzymes required for xylan conversion.

Constructing arabinofuranosidases for dual arabinoxylan debranching activity.

Enzymatic conversion of arabinoxylan requires α-L-arabinofuranosidases able to remove α-L-arabinofuranosyl residues (α-L-Araf) from both mono- and double-substituted D-xylopyranosyl residues (Xylp) in xylan (i.e., AXH-m and AXH-d activity). Herein, SthAbf62A (a family GH62 α-L-arabinofuranosidase with AXH-m activity) and BadAbf43A (a family GH43 α-L-arabinofuranosidase with AXH-d3 activity), were fused to create SthAbf62A_BadAbf43A and BadAbf43A_SthAbf62A. Both fusion enzymes displayed dual AXH-m,d and synergistic activity toward native, highly branched wheat arabinoxylan (WAX). When using a customized arabinoxylan substrate comprising mainly α-(1 → 3)-L-Araf and α-(1 → 2)-L-Araf substituents attached to disubstituted Xylp (d-2,3-WAX), the specific activity of the fusion enzymes was twice that of enzymes added as separate proteins. Moreover, the SthAbf62A_BadAbf43A fusion removed 83% of all α-L-Araf from WAX after a 20 hr treatment. 1 H NMR analyses further revealed differences in SthAbf62A_BadAbf43 rate of removal of specific α-L-Araf substituents from WAX, where 9.4 times higher activity was observed toward d-α-(1 → 3)-L-Araf compared to m-α-(1 → 3)-L-Araf positions.

Laccase/TEMPO oxidation in the production of mechanically strong arabinoxylan and glucomannan aerogels.

New wheat arabinoxylan and konjac glucomannan hydrogels and aerogels were prepared by hemiacetal crosslinking induced by laccase/TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl) -catalysed oxidation, which selectively converts the primary hydroxyl groups to aldehydes. The degree of oxidation of the product aldehydes was ca. 10% of the total carbohydrates of the polysaccharides, and the determination of storage and viscous moduli of the oxidised samples showed that they had formed true hydrogels. Two freezing methods for the hydrogels, conventional freezing and ice crystal templating, were investigated for aerogel production, the ice crystal templated products especially were mechanically strong in compression test against the ice crystals' growth direction. The compressive moduli were ca. 1200kPa for wheat arabinoxylan aerogels and ca. 650kPa for konjac glucomannan aerogels. A morphological study with a scanning electron microscope revealed the inner structure of the aerogels. Ice crystal templated konjac glucomannan aerogel formed round pores with a diameter of ca. 50-100µm. The arabinoxylan aerogel consisted of long and narrow pores with a length of a few hundred µm and width of 50-100µm, which had formed in the direction of the ice crystals' formation. Konjac glucomannan and wheat arabinoxylan are approved food-grade materials, and wheat arabinoxylan is particularly interesting because it can be obtained from cereal processing side streams - thus, these novel products have potential in various applications, including the food, food packaging, and pharmacological fields.

A Novel Colletotrichum graminicola Raffinose Oxidase in the AA5 Family.

We describe here the identification and characterization of a copper radical oxidase from auxiliary activities family 5 (AA5_2) that was distinguished by showing preferential activity toward raffinose. Despite the biotechnological potential of carbohydrate oxidases from family AA5, very few members have been characterized. The gene encoding raffinose oxidase from Colletotrichum graminicola (CgRaOx; EC 1.1.3.-) was identified utilizing a bioinformatics approach based on the known modular structure of a characterized AA5_2 galactose oxidase. CgRaOx was expressed in Pichia pastoris, and the purified enzyme displayed the highest activity on the trisaccharide raffinose, whereas the activity on the disaccharide melibiose was three times lower and more than ten times lower activity was detected on d-galactose at a 300 mM substrate concentration. Thus, the substrate preference of CgRaOx was distinguished clearly from the substrate preferences of the known galactose oxidases. The site of oxidation for raffinose was studied by 1H nuclear magnetic resonance and mass spectrometry, and we confirmed that the hydroxyl group at the C-6 position was oxidized to an aldehyde and that in addition uronic acid was produced as a side product. A new electrospray ionization mass spectrometry method for the identification of C-6 oxidized products was developed, and the formation mechanism of the uronic acid was studied. CgRaOx presented a novel activity pattern in the AA5 family.IMPORTANCE Currently, there are only a few characterized members of the CAZy AA5 protein family. These enzymes are interesting from an application point of view because of their ability to utilize the cheap and abundant oxidant O2 without the requirement of complex cofactors such as FAD or NAD(P). Here, we present the identification and characterization of a novel AA5 member from Colletotrichum graminicola As discussed in the present study, the bioinformatics approach using the modular structure of galactose oxidase was successful in finding a C-6 hydroxyl carbohydrate oxidase having substrate preference for the trisaccharide raffinose. By the discovery of this activity, the diversity of the CAZy AA5 family is increasing.