Cloning and Characterization of a Novel N-Acetyl-D-galactosamine-4-O-sulfate Sulfatase, SulA1, from a Marine Arthrobacter Strain.

Sulfation is gaining increased interest due to the role of sulfate in the bioactivity of many polysaccharides of marine origin. Hence, sulfatases, enzymes that control the degree of sulfation, are being more extensively researched. In this work, a novel sulfatase (SulA1) encoded by the gene sulA1 was characterized. The sulA1-gene is located upstream of a chondroitin lyase encoding gene in the genome of the marine Arthrobacter strain (MAT3885). The sulfatase was produced in Escherichia coli. Based on the primary sequence, the enzyme is classified under sulfatase family 1 and the two catalytic residues typical of the sulfatase 1 family-Cys57 (post-translationally modified to formyl glycine for function) and His190-were conserved. The enzyme showed increased activity, but not improved stability, in the presence of Ca2+, and conserved residues for Ca2+ binding were identified (Asp17, Asp18, Asp277, and Asn278) in a structural model of the enzyme. The temperature and pH activity profiles (screened using p-nitrocatechol sulfate) were narrow, with an activity optimum at 40-50 °C and a pH optimum at pH 5.5. The Tm was significantly higher (67 °C) than the activity optimum. Desulfation activity was not detected on polymeric substrates, but was found on GalNAc4S, which is a sulfated monomer in the repeated disaccharide unit (GlcA-GalNAc4S) of, e.g., chondroitin sulfate A. The position of the sulA1 gene upstream of a chondroitin lyase gene and combined with the activity on GalNAc4S suggests that there is an involvement of the enzyme in the chondroitin-degrading cascade reaction, which specifically removes sulfate from monomeric GalNAc4S from chondroitin sulfate degradation products.

DNA digestion and formation of DNA-network structures with Holliday junction-resolving enzyme Hjc_15-6 in conjunction with polymerase reactions.

The recently identified novel Holliday junction-resolving enzyme, termed Hjc_15-6, activity investigation results imply DNA cleavage by Hjc_15-6 in a manner that potentially enhances the molecular self-assembly that may be exploited for creating DNA-networks and nanostructures. The study also demonstrates Pwo DNA polymerase acting in combination with Hjc_15-6 capability to produce large amounts of DNA that transforms into large DNA-network structures even without DNA template and primers. Furthermore, it is demonstrated that Hjc_15-6 prefers Holliday junction oligonucleotides as compared to Y-shaped oligonucleotides as well as efficiently cleaves typical branched products from isothermal DNA amplification of both linear and circular DNA templates amplified by phi29-like DNA polymerase. The assembly of large DNA network structures was observed in real time, by transmission electron microscopy, on negative stained grids that were freshly prepared, and also on the same grids after incubation for 4 days under constant cooling. Hence, Hjc_15-6 is a promising molecular tool for efficient production of various DNA origamis that may be implemented for a wide range of applications such as within medical biomaterials, catalytic materials, molecular devices and biosensors.

Exploring a novel ß-1,3-glucanosyltransglycosylase, MlGH17B, from a marine Muricauda lutaonensis strain for modification of laminari-oligosaccharides.

The marine environment, contains plentiful renewable resources, e.g. macroalgae with unique polysaccharides, motivating search for enzymes from marine microorganisms to explore conversion possibilities of the polysaccharides. In this study, the first GH17 glucanosyltransglycosylase, MlGH17B, from a marine bacterium (Muricauda lutaonensis), was characterized. The enzyme was moderately thermostable with Tm at 64.4 °C and 73.2 °C, but an activity optimum at 20 °C, indicating temperature sensitive active site interactions. MlGH17B uses ß-1,3 laminari-oligosaccharides with a degree of polymerization (DP) of 4 or higher as donors. Two glucose moieties (bound in the aglycone +1 and +2 subsites) are cleaved off from the reducing end of the donor while the remaining part (bound in the glycone subsites) is transferred to an incoming ß-1,3 glucan acceptor, making a ß-1,6-linkage, thereby synthesizing branched or kinked oligosaccharides. Synthesized oligosaccharides up to DP26 were detected by mass spectrometry analysis, showing that repeated transfer reactions occurred, resulting in several ß-1,6-linked branches. The modeled structure revealed an active site comprising five subsites: three glycone (-3, -2 and -1) and two aglycone (+1 and +2) subsites, with significant conservation of substrate interactions compared to the only crystallized 1,3-ß-glucanosyltransferase from GH17 (RmBgt17A from the compost thriving fungus Rhizomucor miehei), suggesting a common catalytic mechanism, despite different phylogenetic origin, growth environment, and natural substrate. Both enzymes lacked the subdomain extending the aglycone subsites, found in GH17 endo-ß-glucanases from plants, but this extension was also missing in bacterial endoglucanases (modeled here), showing that this feature does not distinguish transglycosylation from hydrolysis, but may rather relate to phylogeny.

A mixture of Nordic berries improves cognitive function, metabolic function and alters the gut microbiota in C57Bl/6J male mice.

Our diets greatly influence our health. Multiple lines of research highlight the beneficial properties of eating berries and fruits. In this study, a berry mixture of Nordic berries previously identified as having the potential to improve memory was supplemented to young C57Bl/6J male mice to investigate effects on cognition function, metabolic health, markers of neuroinflammation, and gut microbiota composition. C57Bl/6J male mice at the age of 8 weeks were given standard chow, a high-fat diet (HF, 60%E fat), or a high-fat diet supplemented with freeze-dried powder (20% dwb) of a mixture of Nordic berries and red grape juice (HF + Berry) for 18 weeks (n = 12 animals/diet group). The results show that supplementation with the berry mixture may have beneficial effects on spatial memory, as seen by enhanced performance in the T-maze and Barnes maze compared to the mice receiving the high-fat diet without berries. Additionally, berry intake may aid in counteracting high-fat diet induced weight gain and could influence neuroinflammatory status as suggested by the increased levels of the inflammation modifying IL-10 cytokine in hippocampal extracts from berry supplemented mice. Furthermore, the 4.5-month feeding with diet containing berries resulted in significant changes in cecal microbiota composition. Analysis of cecal bacterial 16S rRNA revealed that the chow group had significantly higher microbial diversity, as measured by the Shannon diversity index and total operational taxonomic unit richness, than the HF group. The HF diet supplemented with berries resulted in a strong trend of higher total OTU richness and significantly increased the relative abundance of Akkermansia muciniphila, which has been linked to protective effects on cognitive decline. In conclusion, the results of this study suggest that intake of a Nordic berry mixture is a valuable strategy for maintaining and improving cognitive function, to be further evaluated in clinical trials.

Extractability, selectivity, and comprehensiveness in supercritical fluid extraction of seaweed using ternary mixtures of carbon dioxide, ethanol, and water.

It is well-known that an ideal extraction method enabling quantitative analysis should give complete extraction of the target analytes as well as minimal co-extraction of unwanted matrix substances. If the extraction method is part of a nontarget screening protocol, the desired analytes can differ widely in terms of chemical properties. In chromatography, terminologies such as recovery, selectivity, and comprehensiveness are well-established and can easily be determined. However, in extraction, these concepts are much less developed. Hence, the aim of our research is to develop and scrutinize theory in extraction with respect to numerical descriptors for extractability, selectivity, and comprehensiveness. Our approach is based on experiments determining the extractability of target analytes and selected interferences. As a case study, we use a pooled sample of three species of seaweed (Alaria esculenta, Laminaria digitata and Ascophyllum nodosum). Target analytes are ß-carotene, fucoxanthin, δ-tocopherol, and phloroglucinol; and selected interferences are carbohydrates, proteins, ash, arsenic, and chlorophyll a. As a "green and clean" extraction technique, supercritical fluid extraction (SFE) using mixtures of CO2, ethanol and water were explored using a design of experiment. The temperature was varied between 40-80°C, and the pressure was held constant at 300 bar. Obtained results clearly demonstrate that highest relative selectivity was achieved with CO2 containing only 5 vol% of ethanol and no water, which primarily enabled high extractability of ß-carotene, and yielding an extract free of carbohydrates, proteins, and toxic metals such as arsenic. Best methods for highest extractability of the other target analytes varied quite widely. Analytes requiring the highest water content (fucoxanthin and phloroglucinol), also resulted in the lowest relative selectivity. Maximum relative comprehensiveness was achieved using CO2/ethanol/water (40/55/5, v/v/v) at 70°C and 300 bar. Our study demonstrates the feasibility of using relative quantitative descriptors for extractability, selectivity, and comprehensiveness, in optimization strategies for analytical extractions.

Life-cycle assessment of yeast-based single-cell protein production with oat processing side-stream.

Production of fish meal and plant-based feed proteins continues to increase to meet the growing demand for seafood, leading to impacts on marine and terrestrial ecosystems. Microbial proteins such as single-cell proteins (SCPs) have been introduced as feed alternatives since they can replace current fish feed ingredients, e.g., soybean, which are associated with negative environmental impacts. Microbial protein production also enables utilization of grain processing side-streams as feedstock sources. This study assesses the environmental impacts of yeast-based SCP using oat side-stream as feedstock (OS-SCP). Life-cycle assessment with a cradle-to-gate approach was used to quantify global warming, freshwater eutrophication, marine eutrophication, terrestrial acidification, land use, and water consumption of OS-SCP production in Finland. Dried and wet side-streams of oat were compared with each other to identify differences in energy consumption and transportation effects. Sensitivity analysis was performed to examine the difference in impacts at various locations and fermentation times. Benchmarking was used to evaluate the environmental impacts of OS-SCP and other feed products, including both conventional and novel protein products. Results highlight the importance of energy sources in quantifying the environmental performance of OS-SCP production. OS-SCP produced with dried side-streams resulted in higher global warming (16.3 %) and water consumption (7.5 %) than OS-SCP produced from wet side-streams, reflecting the energy and water requirements for the drying process. Compared with conventional products, such as soy protein concentrates, OS-SCP resulted in 61 % less land use, while exacerbating the environmental impacts in all the other categories. OS-SCP had more impact on global warming (205-754 %), water consumption (166-1401 %), freshwater eutrophication (118-333 %), and terrestrial acidification (85-340 %) than other novel products, including yeast protein concentrate, methanotrophic bacterial SCP, and insect meal, while lowering global warming (11 %) and freshwater eutrophication (20 %) compared with dry microalgae biomass.

Novel thermostable GH5_34 arabinoxylanase with an atypical CBM6 displays activity on oat fiber xylan for prebiotic production.

Carbohydrate active enzymes are valuable tools in cereal processing to valorize underutilized side streams. By solubilizing hemicellulose and modifying the fiber structure, novel food products with increased nutritional value can be created. In this study, a novel GH5_34 subfamily arabinoxylanase from Herbinix hemicellulosilytica, HhXyn5A, was identified, produced and extensively characterized, for the intended exploitation in cereal processing to solubilize potential prebiotic fibers: arabinoxylo-oligosaccharides. The purified two-domain HhXyn5A (catalytic domain and CBM6) demonstrated high storage stability, showed a melting temperature Tm of 61°C and optimum reaction conditions were determined to 55°C and pH 6.5 on wheat arabinoxylan. HhXyn5A demonstrated activity on various commercial cereal arabinoxylans and produced prebiotic AXOS, whereas the sole catalytic domain of HhXyn5A did not demonstrate detectable activity. HhXyn5A demonstrated no side activity on oat ß-glucan. In contrast to the commercially available homolog CtXyn5A, HhXyn5A gave a more specific HPAEC-PAD oligosaccharide product profile when using wheat arabinoxylan and alkali extracted oat bran fibers as the substrate. Results from multiple sequence alignment of GH5_34 enzymes, homology modeling of HhXyn5A and docking simulations with ligands XXXA3, XXXA3XX and X5 concluded that the active site of HhXyl5A catalytic domain is highly conserved and can accommodate both shorter and longer ligands. However, significant structural dissimilarities between HhXyn5A and CtXyn5A in the binding cleft of CBM6, due to the lack of important ligand-interacting residues, is suggested to cause the observed differences in substrate specificity and product formation.

Medium development and production of carotenoids and exopolysaccharides by the extremophile Rhodothermus marinus DSM16675 in glucose-based defined media.

BACKGROUND: The marine thermophilic bacterium Rhodothermus marinus can degrade many polysaccharides which makes it interesting as a future cell factory. Progress using this bacterium has, however, been hampered by limited knowledge on media and conditions for biomass production, often resulting in low cell yields and low productivity, highlighting the need to develop conditions that allow studies of the microbe on molecular level. This study presents development of defined conditions that support growth, combined with evaluation of production of carotenoids and exopolysaccharides (EPSs) by R. marinus strain DSM 16675. RESULTS: Two defined media were initially prepared: one including a low addition of yeast extract (modified Wolfe's medium) and one based on specific components (defined medium base, DMB) to which two amino acids (N and Q), were added. Cultivation trials of R. marinus DSM 16675 in shake flasks, resulted in maximum cell densities (OD620 nm) of 2.36 ± 0.057, cell dry weight (CDW) 1.2 ± 0.14 mg/L, total carotenoids 0.59 × 10-3 mg/L, and EPSs 1.72 ± 0.03 mg/L using 2 g/L glucose in DMB. In Wolfe's medium (supplemented by 0.05 g/L yeast extract and 2.5 g/L glucose), maximum OD620 nm was 2.07 ± 0.05, CDW 1.05 ± 0.07 mg/L, total carotenoids 0.39 × 10-3 mg/L, and EPSs 1.74 ± 0.2 mg/L. Growth trials at 5 g/L glucose in these media either failed or resulted in incomplete substrate utilization. To improve reproducibility and increase substrate utilization, a screening of macroelements (e.g. phosphate) in DMB, was combined with use of trace elements and vitamins of the modified Wolfe's medium. The resulting defined minimal R. marinus medium, (DRM), allowed reproducible cultivations to a final OD620nm of 6.6 ± 0.05, CDW 2.85 ± 0.07 mg/L, a maximum specific growth rate (µmax) of 0.26 h-1, total carotenoids 0.77 × 10-3 mg/L and EPSs 3.4 ± 0.17 mg/L in cultivations supplemented with up to 5 g/L glucose. CONCLUSION: A minimal defined medium (DRM) was designed that resulted in reproducible growth and an almost doubled formation of both total carotenoids and EPSs. Such defined conditions, are necessary for systematic studies of metabolic pathways, to determine the specific requirements for growth and fully characterize metabolite production.

Extraction of sugarcane bagasse arabinoxylan, integrated with enzymatic production of xylo-oligosaccharides and separation of cellulose.

Sugarcane processing roughly generates 54 million tonnes sugarcane bagasse (SCB)/year, making SCB an important material for upgrading to value-added molecules. In this study, an integrated scheme was developed for separating xylan, lignin and cellulose, followed by production of xylo-oligosaccharides (XOS) from SCB. Xylan extraction conditions were screened in: (1) single extractions in NaOH (0.25, 0.5, or 1 M), 121 °C (1 bar), 30 and 60 min; (2) 3 × repeated extraction cycles in NaOH (1 or 2 M), 121 °C (1 bar), 30 and 60 min or (3) pressurized liquid extractions (PLE), 100 bar, at low alkalinity (0-0.1 M NaOH) in the time and temperature range 10-30 min and 50-150 °C. Higher concentration of alkali (2 M NaOH) increased the xylan yield and resulted in higher apparent molecular weight of the xylan polymer (212 kDa using 1 and 2 M NaOH, vs 47 kDa using 0.5 M NaOH), but decreased the substituent sugar content. Repeated extraction at 2 M NaOH, 121 °C, 60 min solubilized both xylan (85.6% of the SCB xylan), and lignin (84.1% of the lignin), and left cellulose of high purity (95.8%) in the residuals. Solubilized xylan was separated from lignin by precipitation, and a polymer with ß-1,4-linked xylose backbone substituted by arabinose and glucuronic acids was confirmed by FT-IR and monosaccharide analysis. XOS yield in subsequent hydrolysis by endo-xylanases (from glycoside hydrolase family 10 or 11) was dependent on extraction conditions, and was highest using xylan extracted by 0.5 M NaOH, (42.3%, using Xyn10A from Bacillus halodurans), with xylobiose and xylotriose as main products. The present study shows successful separation of SCB xylan, lignin, and cellulose. High concentration of alkali, resulted in xylan with lower degree of substitution (especially reduced arabinosylation), while high pressure (using PLE), released more lignin than xylan. Enzymatic hydrolysis was more efficient using xylan extracted at lower alkaline strength and less efficient using xylan obtained by PLE and 2 M NaOH, which may be a consequence of polymer aggregation, via remaining lignin interactions.

Novel xylan-degrading enzymes from polysaccharide utilizing loci of Prevotella copri DSM18205.

Prevotella copri is a bacterium that can be found in the human gastrointestinal tract (GIT). The role of P. copri in the GIT is unclear, and elevated numbers of the microbe have been reported both in dietary fiber-induced improvement in glucose metabolism but also in conjunction with certain inflammatory conditions. These findings raised our interest in investigating the possibility of P. copri to grow on xylan, and identify the enzyme systems playing a role in digestion of xylan-based dietary fibers. Two xylan degrading polysaccharide utilizing loci (PUL10 and 15) were found in the genome, with three and eight glycoside hydrolase (GH) -encoding genes, respectively. Three of them were successfully produced in Escherichia coli: One extracellular enzyme from GH43 (subfamily 12, in PUL10, 60 kDa) and two enzymes from PUL15, one extracellular GH10 (41 kDa), and one intracellular GH43 (subfamily 137 kDa). Based on our results, we propose that in PUL15, GH10 (1) is an extracellular endo-1,4-ß-xylanase, that hydrolazes mainly glucuronosylated xylan polymers to xylooligosaccharides (XOS); while, GH43_1 in the same PUL, is an intracellular ß-xylosidase, catalyzing complete hydrolysis of the XOS to xylose. In PUL10, the characterized GH43_12 is an arabinofuranosidase, with a role in degradation of arabinoxylan, catalyzing removal of arabinose-residues on xylan.

Cultivation of the gut bacterium Prevotella copri DSM 18205T using glucose and xylose as carbon sources.

Prevotella copri DSM18205T is a human gut bacterium, suggested as a next-generation probiotic. To utilize it as such, it is, however, necessary to grow the species in a reproducible manner. Prevotella copri has previously been reported to be highly sensitive to oxygen, and hence difficult to isolate and cultivate. This study presents successful batch cultivation strategies for viable strain inoculations and growth in both serum bottles and a stirred tank bioreactor (STR), without the use of an anaerobic chamber, as long as the cells were kept in the exponential growth phase. A low headspace volume in the STR was important to reach high cell density. P. copri utilized xylose cultivated in Peptone Yeast Xylose medium (PYX medium), resulting in a comparable growth rate and metabolite production as in Peptone Yeast Glucose medium (PYG medium) in batch cultivations at pH 7.2.Up to 5 g/L of the carbon source was consumed, leading to the production of succinic acid, acetic acid, and formic acid, and cell densities (OD620 nm ) in the range 6-7.5. The highest yield of produced succinic acid was 0.63 ± 0.05 g/g glucose in PYG medium cultivations and 0.88 ± 0.06 g/g xylose in PYX medium cultivations.

Engineering CGTase to improve synthesis of alkyl glycosides.

Alkyl glycoside surfactants with elongated carbohydrate chains are useful in different applications due to their improved biocompatibility. Cyclodextrin glucanotransferases can catalyze the elongation process through the coupling reaction. However, due to the presence of a hydrophobic tail, the interaction between an alkyl glycoside acceptor and the active site residues is weaker than the interaction with maltooligosaccharides at the corresponding site. Here we report the mutations of F197, G263 and E266 near the acceptor subsites in the CGTase CspCGT13 from Carboxydocella sp. The results showed that substitutions of both F197 and G263 were important for the binding of acceptor substrate dodecyl maltoside during coupling reaction. The double mutant F197Y/G263A showed enhanced coupling activity and displayed a 2-fold increase of the primary coupling product using γ-cyclodextrin as donor when compared to wildtype CspCGT13. Disproportionation activity was also reduced, which was also the case for another double mutant (F197Y/E266A) that however not showed the corresponding increase in coupling. A triple mutant F197Y/G263A/E266A maintained the increase in primary coupling product (1.8-fold increase) using dodecyl maltoside as acceptor, but disproportionation was approximately at the same level as in the double mutants. In addition, hydrolysis of starch was slightly increased by the F197Y and G263A substitutions, indicating that interactions at both positions influenced the selectivity between glycosyl and alkyl moieties.

Engineering the carotenoid biosynthetic pathway in Rhodothermus marinus for lycopene production.

Rhodothermus marinus has the potential to be well suited for biorefineries, as an aerobic thermophile that produces thermostable enzymes and is able to utilize polysaccharides from different 2nd and 3rd generation biomass. The bacterium produces valuable chemicals such as carotenoids. However, the native carotenoids are not established for industrial production and R. marinus needs to be genetically modified to produce higher value carotenoids. Here we genetically modified the carotenoid biosynthetic gene cluster resulting in three different mutants, most importantly the lycopene producing mutant TK-3 (ΔtrpBΔpurAΔcruFcrtB::trpBcrtB T.thermophilus ). The genetic modifications and subsequent structural analysis of carotenoids helped clarify the carotenoid biosynthetic pathway in R. marinus. The nucleotide sequences encoding the enzymes phytoene synthase (CrtB) and the previously unidentified 1',2'-hydratase (CruF) were found fused together and encoded by a single gene in R. marinus. Deleting only the cruF part of the gene did not result in an active CrtB enzyme. However, by deleting the entire gene and inserting the crtB gene from Thermus thermophilus, a mutant strain was obtained, producing lycopene as the sole carotenoid. The lycopene produced by TK-3 was quantified as 0.49 â€‹g/kg CDW (cell dry weight).

Data on saponins, xylan and cellulose yield obtained from quinoa stalks after pressurized hot water extraction.

The data we present below are linked to our research paper "Integrated process for sequential extraction of saponins, xylan and cellulose from quinoa stalks (Chenopodium quinoa Willd.)" (Gil-Ramírez et al., 2018) [1]. The objective is to provide supplementary information in order to facilitate the comprehension of the central composite experimental design (rotatable 22) used in the integrated process of extractions. Two factors, temperature and time of extraction are considered in the design. The responses are the yield of saponin, xylan and cellulose. First, the desirable linear regression obtained by the observed vs. predicted yields plot for each variable response confirm the validation of the model (Fig. 1). Second, the data presented here through Standardized Pareto Charts (Fig. 2), provides information about the effect of the time and temperature, as well as their interactions, in the yield of saponins, xylan and cellulose obtained in an integrated sequential extraction.

ß-Mannanase-catalyzed synthesis of alkyl mannooligosides.

ß-Mannanases catalyze the conversion and modification of ß-mannans and may, in addition to hydrolysis, also be capable of transglycosylation which can result in enzymatic synthesis of novel glycoconjugates. Using alcohols as glycosyl acceptors (alcoholysis), ß-mannanases can potentially be used to synthesize alkyl glycosides, biodegradable surfactants, from renewable ß-mannans. In this paper, we investigate the synthesis of alkyl mannooligosides using glycoside hydrolase family 5 ß-mannanases from the fungi Trichoderma reesei (TrMan5A and TrMan5A-R171K) and Aspergillus nidulans (AnMan5C). To evaluate ß-mannanase alcoholysis capacity, a novel mass spectrometry-based method was developed that allows for relative comparison of the formation of alcoholysis products using different enzymes or reaction conditions. Differences in alcoholysis capacity and potential secondary hydrolysis of alkyl mannooligosides were observed when comparing alcoholysis catalyzed by the three ß-mannanases using methanol or 1-hexanol as acceptor. Among the three ß-mannanases studied, TrMan5A was the most efficient in producing hexyl mannooligosides with 1-hexanol as acceptor. Hexyl mannooligosides were synthesized using TrMan5A and purified using high-performance liquid chromatography. The data suggests a high selectivity of TrMan5A for 1-hexanol as acceptor over water. The synthesized hexyl mannooligosides were structurally characterized using nuclear magnetic resonance, with results in agreement with their predicted ß-conformation. The surfactant properties of the synthesized hexyl mannooligosides were evaluated using tensiometry, showing that they have similar micelle-forming properties as commercially available hexyl glucosides. The present paper demonstrates the possibility of using ß-mannanases for alkyl glycoside synthesis and increases the potential utilization of renewable ß-mannans.

Xylo- and arabinoxylooligosaccharides from wheat bran by endoxylanases, utilisation by probiotic bacteria, and structural studies of the enzymes.

Xylooligosaccharides (XOS) and arabinoxylooligosaccharides (AXOS) were produced from the insoluble arabinoxylan fraction of pretreated wheat bran by endoxylanases. The glycoside hydrolase (GH) family 10 xylanases GsXyn10A from Geobacillus stearothermophilus and RmXyn10A-CM from Rhodothermus marinus produced the AXOS A3X, A2XX and A2 + 3XX in addition to XOS. RmXyn10A-CM also produced XA2 + 3XX due to its non-conserved aglycone region accommodating additional arabinose substitutions in subsite +2. The GH11 enzymes, Pentopan from Thermomyces lanuginosus and NpXyn11A from Neocallimastix patriciarum had minor structural differences affecting hydrogen bonds in subsites -3 and +3, with similar hydrolysis profiles producing XA3XX as major AXOS and minor amounts of XA2XX but different ratios of X3/X2. In vitro analysis of the prebiotic properties of (A)XOS produced by Pentopan revealed nearly complete uptake of X2 and X3 by the probiotic bacteria Lactobacillus brevis and Bifidobacterium adolescentis. In contrast to previous reports, the GH43 arabinofuranosidase BaAXHd-3 from B. adolescentis cleaved α-1,3-linked arabinose on some single substituted AXOS.

Valorization of Brewer's spent grain to prebiotic oligosaccharide: Production, xylanase catalyzed hydrolysis, in-vitro evaluation with probiotic strains and in a batch human fecal fermentation model.

Brewer's spent grain (BSG) accounts for around 85% of the solid by-products from beer production. BSG was first extracted to obtain water-soluble arabinoxylan (AX). Using subsequent alkali extraction (0.5 M KOH) it was possible to dissolve additional AX. In total, about 57% of the AX in BSG was extracted with the purity of 45-55%. After comparison of nine xylanases, Pentopan mono BG, a GH11 enzyme, was selected for hydrolysis of the extracts to oligosaccharides with minimal formation of monosaccharides. Growth of Bifidobacterium adolescentis (ATCC 15703) was promoted by the enzymatic hydrolysis to arabinoxylooligosaccharides, while Lactobacillus brevis (DSMZ 1264) utilized only unsubstituted xylooligosaccharides. Furthermore, utilization of the hydrolysates by human gut microbiota was also assessed in a batch human fecal fermentation model. Results revealed that the rates of fermentation of the BSG hydrolysates by human gut microbiota were similar to that of commercial prebiotic fructooligosaccharides, while inulin was fermented at a slower rate. In summary, a sustainable process to valorize BSG to functional food ingredients has been proposed.

Arabinoxylanase from glycoside hydrolase family 5 is a selective enzyme for production of specific arabinoxylooligosaccharides.

An arabinose specific xylanase from glycoside hydrolase family 5 (GH5) was used to hydrolyse wheat and rye arabinoxylan, and the product profile showed that it produced arabinose substituted oligosaccharides (AXOS) having 2-10 xylose residues in the main chain but no unsubstituted xylooligosaccharides (XOS). Molecular modelling showed that the active site has an open structure and that the hydroxyl groups of all xylose residues in the active site are solvent exposed, indicating that arabinose substituents can be accommodated in the glycone as well as the aglycone subsites. The arabinoxylan hydrolysates obtained with the GH5 enzyme stimulated growth of Bifidobacterium adolescentis but not of Lactobacillus brevis. This arabinoxylanase is thus a good tool for the production of selective prebiotics.

Structural Considerations on the Use of Endo-Xylanases for the Production of prebiotic Xylooligosaccharides from Biomass.

Xylooligosaccharides (XOS) have gained increased interest as prebiotics during the last years. XOS and arabinoxylooligosaccharides (AXOS) can be produced from major fractions of biomass including agricultural by-products and other low cost raw materials. Endo-xylanases are key enzymes for the production of (A)XOS from xylan. As the xylan structure is broadly diverse due to different substitutions, diverse endo-xylanases have evolved for its degradation. In this review structural and functional aspects are discussed, focusing on the potential applications of endo-xylanases in the production of differently substituted (A)XOS as emerging prebiotics, as well as their implication in the processing of the raw materials. Endo-xylanases are found in at least eight different glycoside hydrolase families (GH), and can either have a retaining or an inverting catalytic mechanism. To date, it is mainly retaining endo-xylanases that are used in applications to produce (A)XOS. Enzymes from these GH-families (mainly GH10 and GH11, and the more recently investigated GH30) are taken as prototypes to discuss substrate preferences and main products obtained. Finally, the need of new and accessory enzymes (new specificities from new families or sources) to increase the yield of different types of (A)XOS is discussed, along with in vitro tests of produced oligosaccharides and production of enzymes in GRAS organisms to facilitate use in functional food manufacturing.