3³-α-L-Arabinofuranosyl-xylotetraose (XA³XX)

Understanding the relation between enzyme domain structure and catalytic activity is crucial for optimal engineering of novel enzymes for lignocellulose bioconversion. Xylanases with varying specificities are commonly used to valorise the hemicellulose arabinoxylan (AX), yet characterization of specific arabinoxylanases remain limited. Two homologous GH5_34 arabinoxylanases, HhXyn5A and CtXyn5A, in which the two domains are connected by a 40-residue linker, exhibit distinct activity on AX, yielding different reaction product patterns, despite high sequence identity, conserved active sites and similar domain composition. In this study, the carbohydrate binding module 6 (CBM6), or the inter domain linker together with CBM6, were swapped to investigate their influence on hydrolytic activity and oligosaccharide product pattern on cereal AXs. The variants, with only CBM6 swapped, displayed reduced activity on commercial wheat and rye AX, as well as on extracted oat fibre, compared to the original enzymes. Additionally, exchange of both linker and CBM6 resulted in a reduced ratio of enzyme produced in soluble form in Escherichia coli cultivations, causing loss of activity of both HhXyn5A and CtXyn5A variants. Analysis of oligosaccharide product patterns applying HPAEC-PAD revealed a decreased number of reaction products for CtXyn5A with swapped CBM6, which resembled the product pattern of HhXyn5A. These findings emphasize the importance of the CBM6 interactions with the linker and the catalytic domain for enzyme activity and specificity, and underlines the role of the linker in enzyme structure organisation and product formation, where alterations in linker interactions with the catalytic and/or CBM6 domains, influence enzyme-substrate association and specificity.

This study aimed to enhance the resistance of bread to gastrointestinal digestion by partially substituting wheat flour with starch spherulites. Three types of starch spherulites, specifically A-type (exhibiting an A-type crystalline pattern with mostly positive birefringence), B(−)-type (B-type crystalline with negative birefringence), and B(+)-type (B-type crystalline with positive birefringence), were investigated. The A-, B(−)-, and B(+)-type spherulites showed significantly higher resistant starch contents of 63.5, 63.8, and 89.2 %, respectively, compared to the control wheat flour (7.4 %). The melting temperatures of A-type and B(+)-type spherulites were notably higher than those of the control wheat flour, suggesting the potential preservation of certain enzyme-resistant starch during the baking process. The partial substitution of wheat flour with spherulites resulted in a denser crumb structure, increased bread hardness and chewiness, and a pale brown color in the case of B(+)-type spherulite. However, these variations in physicochemical properties did not significantly impact consumer acceptability. Remarkably, in bread containing A- or B(+)-type spherulite, residual ordered spherulite structures were present after baking, as confirmed by differential scanning calorimetry. This resulted in significantly lower digestibility during in vitro gastrointestinal digestion. These findings are useful for the rational design of bread with sustained glucose release during gastrointestinal digestion.

An uncharacterized gene encoding a glycoside hydrolase family 43-like enzyme from Clostridium boliviensis strain E-1 was identified from genomic sequence data, and the encoded enzyme, CbE1Xyn43-l, was produced in Escherichia coli. CbE1Xyn43-l (52.9 kDa) is a two-domain endo-β-xylanase consisting of a C-terminal CBM6 and a GH43-like catalytic domain. The positions of the catalytic dyad conserved in GH43, the catalytic base (Asp74), and proton donor (Glu240) were identified in alignments including GH43-enzymes of known 3D-structure from different subfamilies. CbE1Xyn43-l is active at pH 7.00-9.0, with optimum temperature at 65°C, and a more than 7 days’ half-life in irreversible deactivation studies at this temperature. The enzyme hydrolyzed birchwood xylan, quinoa stalks glucuronoarabinoxylan, and wheat arabinoxylan with xylotriose and xylotetraose as major hydrolysis products. CbE1Xyn43-l also released xylobiose from pNPX₂ with low turnover (k_cat of 0.044 s⁻¹) but was inactive on pNPX, showing that a degree of polymerization of three (DP3) was the smallest hydrolyzable substrate. Divalent ions affected the specific activity on xylan substrates, which dependent on the ion could be increased or decreased. In conclusion, CbE1Xyn43-l from C. boliviensis strain E-1 is the first characterized member of a large group of homologous hypothetical proteins annotated as GH43-like and is a thermostable endo-xylanase, producing xylooligosaccharides of high DP (xylotriose and xylotetraose) producer.

Background and Objectives: Kilning is crucial in oat processing, to prevent rancidity and extend shelf-life. This study examines kilning effects on the macronutrient composition in Swedish oat varieties Galant, Fatima, and Belinda. We compared two kilning methods: one mimicking industrial practice and a simplified version. We analyzed dietary fibers (arabinoxylan, β-glucan), protein and amino acids, lipid profile, lipase activity, and antioxidant capacity in these oat samples. Findings: Distinct compositional differences were found: Galant had low lipid content, Fatima had elevated lipid and protein levels with fewer carbohydrates, and Belinda was rich in β-glucan and dietary fibers. Both kilning procedures had similar impacts on all varieties, causing no major changes in dietary fiber or total protein content, but resulting in a 20% decrease in soluble proteins. Kilning decreased levels of several amino acids in Belinda, while the l-glutamate/glutamine ratio increased across all varieties. Lipid analysis showed minimal kilning-induced changes; yet, antioxidative capacity diminished. Both kilning methods effectively inactivated lipases. Conclusions: These findings emphasize macronutrient variations among oat varieties and the effect of kilning on soluble proteins, amino acids, and antioxidative capacity.

Cereal arabinoxylans (AXs) are complex polysaccharides in terms of their pattern of arabinose and ferulic acid substitutions, which influence their properties in structural and nutritional applications. We have evaluated the influence of the molecular structure of three AXs from wheat and rye with distinct substitutions on the activity of β-xylanases from different glycosyl hydrolase families (GH 5_34, 8, 10 and 11). The arabinose and ferulic acid substitutions influence the accessibility of the xylanases, resulting in specific profiles of arabinoxylan-oligosaccharides (AXOS). The GH10 xylanase from Aspergillus aculeatus (AcXyn10A) and GH11 from Thermomyces lanuginosus (TlXyn11) showed the highest activity, producing larger amounts of small oligosaccharides in shorter time. The GH8 xylanase from Bacillus sp. (BXyn8) produced linear xylooligosaccharides and was most restricted by arabinose substitution, whereas GH5_34 from Gonapodya prolifera (GpXyn5_34) required arabinose substitution and produced longer (A)XOS substituted on the reducing end. The complementary substrate specificity of BXyn8 and GpXyn5_34 revealed how arabinoses were distributed along the xylan backbones. This study demonstrates that AX source and xylanase specificity influence the production of oligosaccharides with specific structures, which in turn impacts the growth of specific bacteria (Bacteroides ovatus and Bifidobacterium adolescentis) and the production of beneficial metabolites (short-chain fatty acids).

An α-L-arabinofuranosidase (ARF) gene of 1503 bp was synthesized, subcloned into pET26b vector, and expressed in Escherichia coli. The enzyme was purified in active form, and consisted of 500 amino acid residues, corresponding to 55 kD based on SDS-PAGE. The affinity-purified protein was characterized using arabinofuranosyl xylooligosaccharides (AXOS) as substrates. The pH effect was investigated showing an optimum at pH 5.5. XaARF catalyzed the cleavage of arabinose at C3 of the xylopyranosyl unit efficiently if the arabinofuranosyl substitution was at the terminal compared to internal xylose units. The enzyme was able to act on di-substituted xylopyranosyl units with the first cleavage at C3 followed by C2 linkages.

Background: Primary degraders of polysaccharides play a key role in anaerobic biotopes, where plant cell wall accumulates, providing extracellular enzymes to release fermentable carbohydrates to fuel themselves and other non-degrader species. Ruminiclostridium cellulolyticum is a model primary degrader growing amongst others on arabinoxylan. It produces large multi-enzymatic complexes called cellulosomes, which efficiently deconstruct arabinoxylan into fermentable monosaccharides. Results: Complete extracellular arabinoxylan degradation was long thought to be required to fuel the bacterium during this plant cell wall deconstruction stage. We discovered and characterized a second system of “arabinoxylan” degradation in R. cellulolyticum, which challenged this paradigm. This “selfish” system is composed of an ABC transporter dedicated to the import of large and possibly acetylated arabinoxylodextrins, and a set of four glycoside hydrolases and two esterases. These enzymes show complementary action modes on arabinoxylo-dextrins. Two α-L-arabinofuranosidases target the diverse arabinosyl side chains, and two exo-xylanases target the xylo-oligosaccharides backbone either at the reducing or the non-reducing end. Together, with the help of two different esterases removing acetyl decorations, they achieve the depolymerization of arabinoxylo-dextrins in arabinose, xylose and xylobiose. The in vivo study showed that this new system is strongly beneficial for the fitness of the bacterium when grown on arabinoxylan, leading to the conclusion that a part of arabinoxylan degradation is achieved in the cytosol, even if monosaccharides are efficiently provided by the cellulosomes in the extracellular space. These results shed new light on the strategies used by anaerobic primary degrader bacteria to metabolize highly decorated arabinoxylan in competitive environments. Conculsion: The primary degrader model Ruminiclostridium cellulolyticum has developed a “selfish” strategy consisting of importing into the bacterium, large arabinoxylan-dextrin fractions released from a partial extracellular deconstruction of arabinoxylan, thus complementing its efficient extracellular arabinoxylan degradation system. Genetic studies suggest that this system is important to support fitness and survival in a competitive biotope. These results provide a better understanding of arabinoxylan catabolism in the primary degrader, with biotechnological application for synthetic microbial community engineering for the production of commodity chemicals from lignocellulosic biomass.

The application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) in glycan was limited due to their poor ionization efficiency, compared with biomolecules such as proteins and peptides. Aiming to improve the ionization efficiency and simplify preparation procedure simultaneously during MALDI MS analysis, an on-target derivatization method using 3-aminoquinoline (3-AQ)/α-cyano-4-hydroxycinnamic acid (CHCA) as matrix was employed and it was conducted both in the positive and negative ion MALDI TOF MS. Results indicated that after on-target derivatization, the ions generated had substantially improved S/N ratios and sensitivity in the tandem mass spectra. The B/Y- type ions of 3-AQ-labeled glycans could be easily recognized, and cross-ring A- type ions provided additional information to reveal the linkage patterns. Specifically, positive ion mass spectra with protonated adduct as precursor ion produced a simple fragmentation pattern benefited for sequencing and observation of branches. Furthermore, this method was successfully applied in polysaccharides analysis, including arabinoxylan, xylan, arabinogalactan and dextran after enzymatic or acid degradation. This study demonstrated that it was feasible to analyze higher molecular weight polysaccharides by MALDI TOF MS using 3-AQ/CHCA matrix through appropriate hydrolysis, and it allowed much efficient structural interpretation with increased sensitivity and characteristic fragment ions.

The success of establishing bioeconomies replacing current economies based on fossil resources largely depends on our ability to degrade recalcitrant lignocellulosic biomass. This study explores the potential of employing various enzymes acting synergistically on previously pretreated agricultural side streams (corn bran, oat hull, soluble and insoluble oat bran). Degrees of synergy (oligosaccharide yield obtained with the enzyme combination divided by the sum of yields obtained with individual enzymes) of up to 88 were obtained. Combinations of a ferulic acid esterase and xylanases resulted in synergy on all substrates, while a laccase and xylanases only acted synergistically on the more recalcitrant substrates. Synergy between different xylanases (glycoside hydrolase (GH) families 5 and 11) was observed particularly on oat hulls, producing a yield of 57%. The synergistic ability of the enzymes was found to be partly due to the increased enzyme stability when in combination with the substrates.

Arabinoxylan hydrolysates (AXH) are the hydrolyzed products of the major components of the dietary fiber arabinoxylan. AXH include diverse oligosaccharides varying in xylose polymerization and side residue modifications with arabinose at the O-2 and/or O-3 position of the xylose unit. Previous studies have reported that AXH exhibit prebiotic properties on gut bifidobacteria; moreover, several adult-associated bifidobacterial species (e.g., Bifidobacterium adolescentis and Bifidobacterium longum subsp. longum) are known to utilize AXH. In this study, we tried to elucidate the molecular mechanisms of AXH utilization by Bifidobacterium pseudocatenulatum, which is a common bifidobacterial species found in adult feces. We performed transcriptomic analysis of B. pseudocatenulatum YIT 4072^T, which identified three upregulated gene clusters during AXH utilization. The gene clusters encoded three sets of ATP-binding cassette (ABC) transporters and five enzymes belonging to glycoside hydrolase family 43 (GH43). By characterizing the recombinant proteins, we found that three solute-binding proteins of ABC transporters showed either broad or narrow specificity, two arabinofuranosidases hydrolyzed either single- or double-decorated arabinoxylooligosaccharides, and three xylosidases exhibited functionally identical activity. These data collectively suggest that the transporters and glycoside hydrolases, encoded in the three gene clusters, work together to utilize AXH of different sizes and with different side residue modifications. Thus, our study sheds light on the overall picture of how these proteins collaborate for the utilization of AXH in B. pseudocatenulatum and may explain the predominance of this symbiont species in the adult human gut.