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Guar galactomannan (GM) was crosslinked using a sustainable enzymatic oxidation approach to form hydrogels. Nanofibrillated cellulose was used as reinforcement prior to crosslinking. Thirteen solvents were tested for replacing water in the gels, and the volumetric yields of hydrogels are discussed in relation to the solvents’ Hansen solubility parameters. Ethanol and dimethyl sulfoxide (DMSO) were selected for further stepwise solvent exchange, to characterize the hydrogels’ shrinkage in response to solvents at each step. DMSO displayed a good compatibility with GM-based hydrogels as compared to ethanol during stepwise solvent exchange, and the overall shrinkage value was similar with those two solvents after supercritical CO 2 drying. The obtained aerogel exhibited highly porous composite structures with a large surface area (up to 333 m 2 /g) and good mechanical stiffness. Negligible ethanol residue was detected, which makes the aerogels safe materials for food and other life science applications.

Plant-based polysaccharides (cellulose and hemicellulose) are a very interesting option for the preparation of sustainable composite materials to replace fossil plastics, but the optimum bonding mechanism between the hard and soft components is still not well known. In this work, composite films made of cellulose nanofibrils (CNF) and various modified and unmodified polysaccharides (galactoglucomannan, GGM; hydrolyzed and oxidized guar gum, GGhydHox; and guar gum grafted with polyethylene glycol, GG-g-PEG) were characterized from the nano- to macroscopic level to better understand how the interactions between the composite components at nano/microscale affect macroscopic mechanical properties, like toughness and strength. All the polysaccharides studied adsorbed well on CNF, although with different adsorption rates, as measured by quartz crystal microbalance with dissipation monitoring (QCM-D). Direct surface and friction force experiments using the colloidal probe technique revealed that the adsorbed polysaccharides provided repulsive forces-well described by a polyelectrolyte brush model - and a moderate reduction in friction between cellulose surfaces, which may prevent CNF aggregates during composite formation and, consequently, enhance the strength of dry films. High affinity for cellulose and moderate hydration were found to be important requirements for polysaccharides to improve the mechanical properties of CNF-based composites in wet conditions. The results of this work provide fundamental information on hemicellulose-cellulose interactions and can support the development of polysaccharide-based materials for different packaging and medical applications.

Crosslinking-aided gelation was utilized to prepare hydrogels from softwood polysaccharides, with spruce galactoglucomannans (GGM)—a group of largely unexploited hemicelluloses—as the main component, aiming at conversion into sponge-like aerogels. Cellulose nanofibrils were used for the formation of a reinforcing network, which was further crosslinked together with a GGM matrix by ammonium zirconium carbonate, an inorganic salt that is regarded as safe for use in food packaging. The hydrogels were freeze-dried into stiff, low-density aerogels with 98 % of their volume composed of air-filled pores. When immersed in water, the aerogels absorbed water up to 37 times their initial weight, demonstrating elasticity and repeatable and reversible sponge capacity. The developed concept reassembles the wood polysaccharides in a new way, creating interesting possibilities for utilizing the abundant “green gold,” GGM. The obtained biobased materials could find application potential, for example, in the field of food packaging and could contribute in the reduction of the usage of petroleum-based plastics in the future.

Galactose oxidase (EC 1.1.3.9) is a single copper metalloenzyme, having a molecular weight of 65–68 kDa. Galactose oxidase catalyzes the oxidation of primary alcohols to corresponding aldehydes with strict regioselectivity, and the selectivity is high for the galactose C-6 primary hydroxyl group. The oxidation of alcohols to carbonyl compounds is one of the most important reactions in synthetic chemistry, thus enzymatic biocatalysis requiring only molecular oxygen as an oxidant is a valuable alternative to chemical reagents. We review here the various mono-, oligo- and polysaccharide derivatives that have been synthesized by galactose oxidase-catalyzed reactions, and the several either qualitative or quantitative analytical techniques utilized to follow of the reaction, determine the conversion, and identify the products. The optimal reaction conditions, the formation of side products, and the oxidation of substrates other than carbohydrates are discussed. Finally, we summarize engineering efforts of galactose oxidase that have improved recombinant enzyme expression and yield, or altered substrate specificity.

Galactose oxidase (GaO)-catalyzed oxidation of the terminal galactosyl groups of guar galactomannan (GM) and tamarind seed galactoxyloglucan (XG) results in cross-linking of these polysaccharides via hemiacetal bonds and formation of elastic hydrogels (GMox and XGox, respectively), and enables the formation of aerogels. Nanofibrillated cellulose (NFC) was added at varying quantities to the aqueous GM and XG solutions and was entrapped in the three-dimensional structure of polysaccharides by enzyme mediated gelation. Addition of NFC up to 25% did not hinder the enzyme activity. The reinforcing effect of NFC on hydrogels was measured with rheometer and texture analyzer. The GMox hydrogels with 25% NFC exhibited higher elastic modulus than corresponding XGox hydrogels, but the latter showed higher compressive modulus. On the other hand, the lyophilized GMox aerogels containing 25% NFC showed the highest compressive modulus, 167 kPa. The reinforcing effect of NFC depended on the type of polysaccharides (GM/XG) and state of the material (hydro- or aerogel). Viewing the aerogels with focused ion beam scanning electron microscopy showed that the pores were approximately 125–250 μm in diameter.

All-polysaccharide composite films were prepared from native, unmodified cellulose nanofibrils (CNF) mixed with various natural water-soluble polysaccharides like carboxymethyl cellulose, galactoglucomannan, xyloglucan and guar gum. Composite films were manufactured by pressurized filtration and hot pressing. The mechanical properties of the films were systematically evaluated in the dry and the wet state. GG was furthermore selectively oxidized using galactose oxidase (EC 1.1.3.9), and the effect of the degree of oxidation on the final composite film properties was shown. It was found that all the tested polysaccharides increased the strength and toughness of the dry composite films at 2 weight percent (wt.%) addition to CNF. After soaking the samples for 24 h in water, striking differences between the samples were found: already at 2 wt.% CMC the wet strength of the composite films diminished, while the uncharged polysaccharides improved the wet strength. For example, the addition of 2 wt.% GGM increased Young's modulus by a factor of 1.3, the tensile strength by a factor of 2.8, and the toughness by a factor of 3.4. The results are discussed in relation to the amount of water absorbed in the films and possible reasons for the improved properties are suggested.

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.

Galactose units of spruce galactoglucomannan (GGM), guar galactomannan (GM), and tamarind (galacto)xyloglucan (XG) were selectively allylated. Firstly aldehyde functionalities were formed at the C-6 position via enzymatic oxidation by galactose oxidase. The formed aldehydes were further derivatized by an indium mediated Barbier-Grignard type reaction, resulting in the formation of homoallylic alcohols. In addition to allylic halides, the same reaction procedure was also applicable for GGM, when using propargyl bromide as halide. All reaction steps were done in water, thus the polysaccharides were modified in a one-pot reaction. The formation of the allylated, or propargylated, product was identified by MALDI-TOF-MS. All polysaccharide products were isolated and further characterized by GC-MS or NMR spectroscopy. By this chemo-enzymatic process, we have demonstrated a novel method for derivatization of GGM and other galactose-containing polysaccharides. The derivatized polysaccharides are potential platforms for further functionalizations.

Aerogels are porous and lightweight materials capable of active sorption and releasing desired compounds and/or bearing mechanical load. Using polysaccharides as aerogel matrices instead of silicate or synthetic polymers has additional benefits. Polysaccharides can be developed as bio-based, biodegradable, and/or edible materials in food applications. Polysaccharides that could be used to prepare aerogels include cellulose, hemicelluloses, marine polysaccharides, and starch, all of which have in common the ability to form gels either by themselves in the presence of water or with dications, other cross-linking agents, and/or other, blended, or mixed polysaccharides. After the liquid phase is removed, the dry aerogels form solid particles of various shapes and sizes that have high porosity and surface area. These characteristics indicate vast possibilities for the use of polysaccharide aerogels as advanced food materials.

Carboxylated, anionic polysaccharides were selectively prepared using a combination of enzymatic and chemical reactions. The galactose-containing polysaccharides studied were spruce galactoglucomannan, guar galactomannan, and tamarind galactoxyloglucan. The galactosyl units of the polysaccharides were first oxidized with galactose oxidase (EC 1.1.3.9) and then selectively carboxylated, resulting in the galacturonic acid derivatives with good conversion and yield. The degrees of oxidation (DO) of the products were determined by gas chromatography-mass spectrometry (GC-MS). A novel feasible electrospray ionization-mass spectrometry (ESI-MS) method was also developed for the determination of DO. The solution properties and charge densities of the products were investigated. The interaction of the products with cellulose was studied by two methods, bulk sorption onto bleached birch kraft pulp and adsorption onto nanocellulose ultrathin films by quartz crystal microbalance with dissipation (QCM-D). To study the effect of the location of the carboxylic acid groups on the physicochemical properties, polysaccharides were also oxidized by 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-mediated reaction producing polyuronic acids. The chemo-enzymatically oxidized galacturonic polysaccharides with an unmodified backbone had a better ability to interact with cellulose than the TEMPO-oxidized products. The selectively carboxylated polysaccharides can be further exploited, as such, or in the targeted functionalization of cellulose surfaces.

Polysaccharides guar galactomannan (guar gum), locust bean galactomannan (locust bean gum) and tamarind galactoxyloglucan were selectively oxidized by galactose oxidase. The degrees of oxidation of the products were 18–28% for guar galactomannan, 10–16% for locust bean galactomannan and 12–14% for tamarind galactoxyloglucan, calculated from the ratio of oxidized galactose units and total carbohydrates. The rheological properties of the unoxidized and oxidized polysaccharide solutions were investigated by determining their viscosities, storage and loss moduli, and temperature dependence of moduli from 20 °C to 90 °C. All the studied oxidized polysaccharides formed hydrogels throughout the entire temperature range. Concentration (0.2–1% w/v) and degree of oxidation had an effect on the gel formation. The oxidized galactomannans formed stable gels already in low concentrations, such as 0.2–0.4% w/v, while oxidized galactoxyloglucan required a concentration of 0.8% w/v to be stable up to 90 °C. The oxidized polysaccharide hydrogels are highly potential materials for food and medical applications requiring thermal stability.

The C-6 unit of methyl α-D-galactopyranoside was selectively modified by combining enzymatic oxidation with an indium-mediated allylation reaction. The Barbier-Grignard type reaction, where a carbonyl group reacts with an allyl halide, proceeds in aqueous solution, even with water as the only solvent; thus carbohydrates can be modified without the need for drying or protection-deprotection steps. The corresponding homoallyl alcohols are produced in high yields of >90% in the reactions with allyl bromide and cinnamyl chloride. The main products were isolated and characterized by GC-MS and NMR spectroscopy.

Galactose oxidase was used as a catalyst to oxidize selectively the C-6 hydroxyls of terminal galactose to carbonyl groups. The polysaccharides studied included spruce galactoglucomannan, guar galactomannan, larch arabinogalactan, corn fiber arabinoxylan, and tamarind seed xyloglucan, with terminal galactose contents varying from 6% to 40%. A multienzyme system was used, with catalase and horseradish peroxidase to enhance the action of galactose oxidase. An analysis technique was developed for the quantification of the reactive aldehydes with GC-MS, utilizing NaBD4 reduction and acidic methanolysis. The best oxidation degrees of terminal galactosyls were obtained with xyloglucan (85% of galactose) and spruce galactoglucomannan (65% of galactose). The highest oxidation degree based on total carbohydrates was achieved with guar gum (28%), which had the highest galactose content. The oxidation resulted in changes in the physicochemical properties of the polysaccharide solutions, and the changes observed varied between the polysaccharides. The clearest change was in tamarind xyloglucan, which formed a gel after the oxidation. After the oxidation, larger particles were present in the solution of spruce galactoglucomannan, but changes in its rheological properties were not observed.

Four alternative strategies for the preparation of deuterium poly-labelled 5-n-alkylresorcinols are explored. Ring-labelled 2H3-alkylresorcinols synthesized by acidic H/D exchange are stable under electrospray ionization MS conditions but scrambling occurs in electron bombardment ionization MS. Side chain-labelled 2H4-derivatives prepared by two different total synthesis approaches are contaminated by isotopologues with varying number of deuterium labels due to H/D redistribution and exchange during D2 gas deuterogenation. The derivative carrying an ω-2H3 label is isotopically pure and completely stable under all relevant analytical conditions encountered in quantitation work. Copyright © 2008 John Wiley & Sons, Ltd.

openaire: EC/H2020/33947/EU//BHIVE Background Galactose oxidase (GaO) selectively oxidizes the primary hydroxyl of galactose to a carbonyl, facilitating targeted chemical derivatization of galactose-containing polysaccharides, leading to renewable polymers with tailored physical and chemical properties. Here we investigate the impact of a family 29 glucomannan binding module on the activity and binding of GaO towards various polysaccharides. Specifically, CBM29-1-2 from Piromyces equi was separately linked to the N- and C-termini of GaO. Results Both GaO-CBM29 and CBM29-GaO were successfully expressed in Pichia pastoris, and demonstrated enhanced binding to galactomannan, galactoglucomannan and galactoxyloglucan. The position of the CBM29 fusion affected the enzyme function. Particularly, C-terminal fusion led to greatest increases in galactomannan binding and catalytic efficiency, where relative to wild-type GaO, kcat/Km values increased by 7.5 and 19.8 times on guar galactomannan and locust bean galactomannan, respectively. The fusion of CBM29 also induced oligomerization of GaO-CBM29. Major conclusions Similar to impacts of cellulose-binding modules associated with cellulolytic enzymes, increased substrate binding impeded the action of GaO fusions on more concentrated preparations of galactomannan, galactoglucomannan and galactoxyloglucan; this was especially true for GaO-CBM29. Given the N-terminal positioning of the native galactose-binding CBM32 in GaO, the varying impacts of N-terminal versus C-terminal fusion of CBM29-1-2 may reflect competing action of neighboring CBMs. General significance This study thoroughly examines and discusses the effects of CBM fusion to non-lignocellulytic enzymes on soluble polysaccharides. Herein kinetics of GaO on galactose containing polysaccharides is presented for the first time.

I. E. Marko [Universite catholique de Louvain, Belgium]—Novel Orthoesters in Organic Synthesis. S. J. Harwood [GlaxoSmithKline, UK]—The Synthesis of Stable Labelled Ketamine. Th. Moenius, R. Salter, B. Wietfeld, P. Burtscher, C. Zueger [Novartis, Switzerland]—C-13, C-14, N-15 Labelling of the 4-Cyanobenzyl bromide – A Versatile Labelling Building Block. J. Clayden [University of Manchester, UK]—Synthesis and Stereocontrol with Lithiated Amides. R. Salter, K. Bordeaux, P. Burtscher, Y. Metz, Th. Moenius, I. Rodriguez, R. Ruetsch, R. Voges, C. Zueger [Novartis, Switzerland]—Isotopic Labelling of STI571 and its Metabolites in the Development of Gleevec®. J. M. Gardiner, W. Stimpson, N. Panchal [University of Manchester, UK], J. Herbert, G. J. Ellames [Sanofi-Aventis, UK]—Synthesis of Labelled Carbohydrates. M. Beller [Rostock University, Germany]—Homogeneous Catalysis – A Powerful Tool for the Synthesis of Pharmaceuticals. J. Kozempel, J. Kadeřavek, L. Lesetický, O. Lebeda [University of Prague, Czech]—Preparation of Ortho-radiohalogenated Phenylazides and Triazenes, Versatile Building Blocks for Indirect Labelling. K. Wahala, P. Kiuru, E. Leppala, M. Pohjoispaa, K. Parikka, B. Raffaelli [University of Helsinki, Finland]—Controlled Polydeuteration of Estrogenic Compounds. J. M. Herbert [Sanofi-Aventis, UK]—Catalyst Stability in Iridium-mediated Isotope Exchange. C. G. M. Janssen, W.L.M. Verluyten, M. Vliegen [Johnson & Johnson Pharmaceutical, Belgium]—Pd/C Catalysed Hydrogenolytic H/T Exchange in Solvents, Effects on Solvents and Product in a Bromine-tritium Exchange Reaction. P. Mading, F. Fuchtner, R. Bergmann, J. Pietzsch, C. Hultsch, F. Wust [Forschungszentrum Rossendorf, Germany]—A New Module-Assisted Synthesis of the Versatile, Bifunctional Labelling Agent [18F]SFB: From Radiochemistry to Applications. M. Scheunemann, J. Vercouillie, St. Fischer, J. Steinbach [Institut fur Interdisziplinare Isotopenforschung Leipzig, Germany], D. Sorger, U. Grosmann, O. Sabri, R. Schliebs [University Leipzig, Germany]—Syntheses and First Biological Evaluation of 18F Labelled Bicyclic Analogues of Vesamicol as Potential VAChT Imaging Agents.

The first synthesis of bioactive long alkyl chain 5-n-alkylresorcinols, present in whole grain products, by a novel modification of the Wittig reaction is described. All the main long chain 5-n-alkylresorcinols present in rye and wheat, including C-23 and C-25 analogues and haptens, which have not been previously prepared, were synthesised. Microwave-promoted reactions of a semi-stabilized ylid and alkanals in water gave good yields in both pressurized and open systems. An alternative microwave-promoted synthesis starting from non-stabilized alkyltriphenylphosphonium salts and 3,5-dimethoxybenzaldehyde worked as well. Aqueous media were suitable for the reactions even if the starting materials were not soluble in water. The 5-n-alkylresorcinols are potential biomarkers of whole grain intake, and the new hapten derivatives of 5-n-alkylresorcinols will open the way for the immunochemical detection techniques of alkylresorcinols. The first synthesis of bioactive long alkyl chain 5-n-alkylresorcinols, present in whole grain products, by a novel modification of the Wittig reaction is described. All the main long chain 5-n-alkylresorcinols present in rye and wheat, including C-23 and C-25 analogues and haptens, which have not been previously prepared, were synthesised. Microwave-promoted reactions of a semi-stabilized ylid and alkanals in water gave good yields in both pressurized and open systems. An alternative microwave-promoted synthesis starting from non-stabilized alkyltriphenylphosphonium salts and 3,5-dimethoxybenzaldehyde worked as well. Aqueous media were suitable for the reactions even if the starting materials were not soluble in water. The 5-n-alkylresorcinols are potential biomarkers of whole grain intake, and the new hapten derivatives of 5-n-alkylresorcinols will open the way for the immunochemical detection techniques of alkylresorcinols. The first synthesis of bioactive long alkyl chain 5-n-alkylresorcinols, present in whole grain products, by a novel modification of the Wittig reaction is described. All the main long chain 5-n-alkylresorcinols present in rye and wheat, including C-23 and C-25 analogues and haptens, which have not been previously prepared, were synthesised. Microwave-promoted reactions of a semi-stabilized ylid and alkanals in water gave good yields in both pressurized and open systems. An alternative microwave-promoted synthesis starting from non-stabilized alkyltriphenylphosphonium salts and 3,5-dimethoxybenzaldehyde worked as well. Aqueous media were suitable for the reactions even if the starting materials were not soluble in water. The 5-n-alkylresorcinols are potential biomarkers of whole grain intake, and the new hapten derivatives of 5-n-alkylresorcinols will open the way for the immunochemical detection techniques of alkylresorcinols.

The reaction conditions of galactose oxidase-catalyzed, targeted C-6 oxidation of galactose derivatives were optimized for aldehyde production and to minimize the formation of secondary products. Galactose oxidase, produced in transgenic Pichia pastoris carrying the galactose oxidase gene from Fusarium spp., was used as catalyst, methyl alpha-D-galactopyranoside as substrate, and reaction medium, temperature, concentration, and combinations of galactose oxidase, catalase, and horseradish peroxidase were used as variables. The reactions were followed by (1)H NMR spectroscopy and the main products isolated, characterized, and identified. An optimal combination of all the three enzymes gave aldehyde (methyl alpha-D-galacto-hexodialdo-1,5-pyranoside) in approximately 90% yield with a substrate concentration of 70 mM in water at 4 degrees C using air as oxygen source. Oxygen flushing of the reaction mixture was not necessary. The aldehyde existed as a hydrate in water. The main secondary products, a uronic acid (methyl alpha-D-galactopyranosiduronic acid) and an alpha,beta-unsaturated aldehyde (methyl 4-deoxy-alpha-D-threo-hex-4-enodialdo-1,5-pyranoside), were observed for the first time to form in parallel. Formation of uronic acid seemed to be the result of impurities in the galactose oxidase preparation. (1)H and (13)C NMR data of the products are reported for the alpha,beta-unsaturated aldehyde for the first time, and chemical shifts in DMSO-d(6) for all the products for the first time. Oxidation of D-raffinose (alpha-D-galactopyranosyl-(1-6)-alpha-D-glucopyranosyl-(1-2)-beta-D-fructofuranoside) in the same optimum conditions also proceeded well, resulting in approximately 90% yield of the corresponding aldehyde.

Alkylresorcinols can be found in high amounts in whole grain cereals, especially in rye. Previously it has not been possible to measure alkylresorcinols in plasma. In this paper a validated gas chromatographic–mass spectrometric method for the quantitative determination of alkylresorcinols with chain lengths of C15:0, C17:0, C19:0, C21:0, and C23:0 in human plasma samples is presented. Other alkylresorcinols may be measured with the method as well, but their assay was not validated in this work. In this work also the amount of alkylresorcinol C25:0 was measured. The pretreatment of plasma samples consists of a simple incubation, an extraction with diethyl ether and a chromatographic purification before the GC-MS analysis. As internal standard an alkylresorcinol C20:0 was used. The validation of the method showed that it fulfilled the reliability criteria. Calibration graphs were linear over the range of 4.1–660 pg per injection. The mean recovery percentage was 112±10.8%. Our results show that the alkylresorcinols are found in plasma in the same ratio, as found in rye grains, according to literature. The alkylresorcinols were in the unconjugated form. The total amounts of alkylresorcinols in two plasma samples analyzed here were 333 and 381 nmol/L.

Isotopically and isomerically pure polydeuterated flavonoids and isoflavonoids have been prepared for quantitation of these compounds in biological matrices. Various deutero-labeling techniques are presented and methods for establishing the isotopical and isomerical purity of deuterated products are discussed.

Specific enzymatic oxidation of terminal galactosyl-containing polysaccharides (guar galactomannan (GM), and tamarind seed galactoxyloglucan (XG)) was used to prepare hydrogels. The hydrogels were lyophilized to form novel types of polysaccharide aerogels: biobased and biodegradable, lightweight, and stiff materials. The compressive moduli of the aerogels were greatly dependent on the oxidation, polysaccharide type, freezing method, and ambient moisture. Ice crystal templated, oriented aerogels from oxidized XG (XG-OX) showed the highest compressive modulus, 359 kPa, when determined parallel to the freezing and drying direction (i.e., the vertical direction). The water vapor sorption of freeze-dried GM and XG was not significantly affected by oxidation, even though the oxidized GM (GM-OX) and XG-OX aerogels were no longer water-soluble. GM-OX and XG-OX aerogels absorbed liquid water 40 and 20 times their initial weight, respectively. Focused ion beam scanning electron microscopy showed that the inner structure of oriented aerogels from GM-OX consisted of a honeycomb architecture with a pore diameter of some tens of micrometers. On the other hand, corresponding aerogels from XG-OX seemed to contain longer capillaries oriented in the freezing direction. This observation was supported by imaging the XG-OX aerogels using high-resolution synchrotron X-ray microtomography. The enzymatic hydro- and aerogel preparation method is considered a green way to obtain novel, functional products from polysaccharides.

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.