Rhamnogalacturonan (Soy Bean)

Arabinoxylan is a prevalent hemicellulose type, notably heterogeneous and resistant to biodegradation. Arabinofuranosidases (Abfs) remove arabinofuranosyl decorations of arabinoxylan, thus enabling hydrolysis by xylanases. However, a variety of Abf and xylanase specificities have emerged in recent years, necessitating a deeper understanding of their role in biomass degradation. This work investigates the biochemical features of TtAbf43 from Thermothelomyces thermophila, which specifically removes the O-3-linked arabinofuranose moieties from di-substituted xylopyranoses. The enzyme also exhibited secondary hydrolytic activity on o-nitrophenyl-β-d-xylopyranoside and arabinan. The hydrolysis of pretreated wheat and corn bran substrates was assessed using TtAbf43 and AnAbf51, two enzymes with distinct catalytic specificities. The Abfs enhanced the performance of endo-xylanases TmXyn10 and AnXyn11, promoting the release of xylo-oligomers, while the xylanases, in turn, stimulated arabinose release by the Abfs. Additionally, the Abfs facilitated the endo- and exo-activities of the bifunctional xylobiohydrolase/glucuronoxylanase TtXyn30A for the release of xylobiose and short aldouronic acids from biomass. AnAbf51 also acted in synergy with the acetyl xylan esterase OCE6 and the exo-deacetylase TtCE16B in debranching enzymatically derived oligomers from lignocellulose, whereas TtAbf43 remained unaffected by the esterases. These diverse synergistic relationships among different hemicellulases could assist the development of new enzymatic approaches for efficient biomass valorization.

Considering an ever-growing global population, which hit 8 billion people in the fall of 2022, it is essential to find solutions to avoid croplands competition between human food and animal feed. Agricultural co-products such as soybean meals have become important components of the circular economy thanks to their use in animal feed. Their implementation was made possible by the addition of exogenous enzymes in the diet of monogastric animals, especially fungal carbohydrate-active enzymes (CAZymes). Here, we describe a time-course production and analysis of Aspergillus terreus secretomes for the identification of CAZymes able to enhance the digestibility of soybean meals. Functional assays revealed that the release of nutrients and the degradation of pectins in soybean meals can be tightly interconnected. Using a comparative proteomics approach, we identified several fungal pectin-degrading enzymes leading to increased assimilable nutrients in the soluble fraction of soybean meals. Our results reinforce the importance of deconstructing pectic polysaccharides in feedstuffs and contribute to sharpen our understanding of the fungal enzymatic interplays involved in pectin hydrolysis.IMPORTANCEIn the present study, we developed a strategy to identify the key fungal enzymatic activities involved in the improvement of soybean meal (SBM) digestibility. Our data unravel the importance of pectin degradation for the release of nutrients from SBM and provide some insights regarding the degradation of rhamnogalacturonan-I (RG-I) by ascomycetes. Indeed, the hydrolysis of pectins and RG-I by human microbiota is well documented in the literature, but our knowledge of the fungal CAZymes at play for the degradation of soybean pectins remains hitherto underexplored. Due to its wide use in animal feed, improving the digestibility of SBM by enzymatic treatments is a current challenge for feed additive suppliers. Since non-starch polysaccharides and pectins have often been reported for their anti-nutritional role in SBM, we believe this study will provide new avenues toward the improvement of enzymatic cocktails for animal nutrition and health.

Fungal fermentation of food and agricultural by-products holds promise for improving food sustainability and security. However, the molecular basis of fungal waste-to-food upcycling remains poorly understood. Here we use a multi-omics approach to characterize oncom, a fermented food traditionally produced from soymilk by-products in Java, Indonesia. Metagenomic sequencing of samples from small-scale producers in Western Java indicated that the fungus Neurospora intermedia dominates oncom. Further transcriptomic, metabolomic and phylogenomic analysis revealed that oncom-derived N. intermedia utilizes pectin and cellulose degradation during fermentation and belongs to a genetically distinct subpopulation associated with human-generated by-products. Finally, we found that N. intermedia grew on diverse by-products such as fruit and vegetable pomace and plant-based milk waste, did not encode mycotoxins, and could create foods that were positively perceived by consumers outside Indonesia. These results showcase the traditional significance and future potential of fungal fermentation for creating delicious and nutritious foods from readily available by-products.

Dietary oligo- and polysaccharides modulate gut microbiota and thus exert prebiotic activity, which is determined by their heterogeneous structure. To explore the correlations between monosaccharide profile and microbial community, simulated gut fermentation of different glycans, including arabinan (ArB), galactooligosaccharide (GOS), arabinogalactan (ArG), rhamnogalacturonan (RhG), and xyloglucan (XyG) that are characterized by typical sugar residues were performed. Results showed that RhG displayed high contents of galacturonic acid (344.79 mg/g), rhamnose (171.70 mg/g), and galactose (151.77 mg/g), and the degradation ratio of them after fermentation was 73.87 %, 84.96 %, and 87.11 %, respectively. Meanwhile, the relative abundance of glycan-degrading bacteria Bacteroides in the RhG was boosted from 4 h (4.97 %) to 48 h (36.45%). Butyrate-generating bacteria Megasphaera (56.69 %) and Bifidobacterium (28.02 %) are dominant genera in the ArB, which generated the highest concentration of carbohydrate-metabolite (94.58 mmol/L) in terms of acetate, propionate, butyrate and valerate, followed by the ArG (87.36 mmol/L). However, ammonia generation of the ArG increased rapidly, representing the highest content of protein-metabolite (66.36 mmol/L) including ammonia, isobutyrate, and isovalerate. As compared, metabolites generated from protein and carbohydrates grow steadily at a low level during the XyG fermentation. Correlation analysis further indicated that Bacteroides was positively correlated with propionate (p < 0.001), galacturonic acid (p < 0.001), and rhamnose (p < 0.05), while Bifidobacterium has positive correlation with butyrate and arabinose (p < 0.01). Overall, monosaccharides composition in the different oligo- and polysaccharides induces distinct responses of the dominant microbiota and thus modulates the subsequent fermentation metabolites of carbohydrate and protein, promoting a deep understanding of the structure-fermentation relationship of dietary glycans.

The impact of pectin structure on carotenoid bioaccessibility is still uncertain. This study aims to investigate how the different pectic polymers affected the bioaccessibility of carotenoids in a simulated juice model during static in vitro digestion. This study includes homogalacturonan (HG), which is a linear pectic polymer, rhamnogalacturonan-I (RG-I), which is a branched pectic polymer, and rhamnogalacturonan (RG), which is a diverse pectic polymer rich in RG-I, rhamnogalacturonan-II (RG-II), and xylogalacturonan domains. Juice models without pectin had the highest carotenoid bioaccessibility, suggesting pectin has negative effects on carotenoid bioaccessibility. During the intestinal phase, systems with HG showed the highest viscosity, followed by systems with RG and systems with RG-I. Systems with RG-I had lower carotenoid bioaccessibility than systems with HG and RG-II. Both the percentage of RG-I and the average side chain length of RG-I had negative correlations with carotenoid bioaccessibility. RG-I side chains with more arabinose and/or galactose might cause lower carotenoid bioaccessibility in this juice model system. This study offers valuable insights into the relationship between pectin structure and carotenoid bioaccessibility in a simulated juice model, highlighting the importance of considering pectin composition for maximizing carotenoid bioaccessibility and potential health benefits in fruit-based beverages.

Rhamnogalacturonans are branched pectic polysaccharides found in most plant cell walls and various agro-industrial residues. A commercial rhamnogalacturonan from soybean was selected as a model substrate to investigate the hydrolytic capacity of aqueous carboxylic acids (citric and malic acids) under subcritical water conditions as a green approach to biomass valorization. The hydrolysis was performed in batch mode at different temperatures (125-155°C) and reaction times (10-120 min) at constant pressure (100 bar). The HPSEC-RID and HILIC-ELSD analyses showed that aqueous carboxylic acids at 125°C/100 bar favored cleavage of neutral sugar residues from side chains of rhamnogalacturonan. At 135°C/100 bar/60 min, scission of rhamnogalacturonan backbone was evident where fractions of 4.7 kDa, 2.1 kDa, and <1.4 kDa were prevalent. Fractions with ≤2.1 kDa were comprised of 2-9 DP (degree of polymerization) oligogalacturonides and 5-10 DP galacto-oligosaccharides. A multi-step sequential hydrolysis mechanism was proposed for rhamnogalacturonan hydrolysis in subcritical water-carboxylic acid media.

Three recombinant β-galactosidases (BGALs; PcBGAL35A, PcBGAL35B, and PcGALX35C) belonging to the glycoside hydrolase (GH) family 35 derived from Penicillium chrysogenum 31B were expressed using Pichia pastoris and characterized. PcBGAL35A showed a unique substrate specificity that has not been reported so far. Based on the results of enzymological tests and ¹H-nuclear magnetic resonance, PcBGAL35A was found to hydrolyze β-1,4-galactosyl residues linked to L-rhamnose in rhamnogalacturonan-I (RG-I) of pectin, as well as p-nitrophenyl-β-D-galactopyranoside and β-D-galactosyl oligosaccharides. PcBGAL35B was determined to be a common BGAL through molecular phylogenetic tree and substrate specificity analysis. PcGALX35C was found to have similar catalytic capacities for the β-1,4-galactosyl oligomer and polymer. Furthermore, PcGALX35C hydrolyzed RG-I-linked β-1,4-galactosyl oligosaccharide side chains with a degree of polymerization of 2 or higher in pectin. The amino acid sequence similarity of PcBGAL35A was approximately 30% with most GH35 BGALs, whose enzymatic properties have been characterized. The amino acid sequence of PcBGAL35B was approximately 80% identical to those of BGALs from Penicillium sp. The amino acid sequence of PcGALX35C was classified into the same phylogenetic group as PcBGAL35A. Pfam analysis revealed that the three BGALs had five domains including a catalytic domain. Our findings suggest that PcBGAL35A and PcGALX35C are enzymes involved in the degradation of galactosylated RG-I in pectin. The enzymes characterized in this study may be applied for products that require pectin processing and for the structural analysis of pectin.

Pectin oligosaccharides (POSs) are being recognized as potent prebiotics, given their structural complexity. Here, POSs were generated from apple pectin, rhamnogalacturonan-I and homogalacturonan with different degrees of esterification (high and low) using an optimized concentration of trifluoroacetic acid. The resulting POSs were analytically characterized, revealing that they contain linear and branched oligomers with a degree of polymerization (DP) up to 7. Biological activity of the generated POSs was determined in terms of immuno- and bacterio-modulatory perspectives. POSs significantly reduced the inflammatory response triggered by lipopolysaccharide and promoted the growth of several bacteria beneficial to the human gut. Overall results indicate that the degree of esterification of POSs is not a key element, but length of the DP and structure of POSs are responsible for biological outcomes. Owing to their biological activity, the POSs generated here can be considered as effective prebiotics and can be exploited for maintaining immune and microbial homeostasis in the human gut.

Exo‐rhamnogalacturonan lyase from Penicillium chrysogenum 31B (PcRGLX) was recently classified as a member of polysaccharide lyase (PL) family 26 along with hypothetical proteins derived from various organisms. In this study, we determined the crystal structure of PcRGLX as the first structure of a member of this family. Based on the substrate‐binding orientation and substrate specificity, PcRGLX is an exo‐type PL that cleaves rhamnogalacturonan from the reducing end. Analysis of PcRGLX‐complex structures with reaction products indicate that the active site possesses an L‐shaped cleft that can accommodate galactosyl side chains, suggesting side‐chain‐bypassing activity in PcRGLX. Furthermore, we determined the residues critical for catalysis by analyzing the enzyme activities of inactive variants.

The primary plant cell wall is composed of a complex network of pectin, hemicellulose and cellulose. Potential interactions between these polysaccharides were studied for carrot, tomato and strawberry, with a focus on the role of pectin. The Chelating agent Unextractable Solids (ChUS), the residue after water- and EDTA extraction, was ball milled and subsequently water extracted. For tomato and strawberry, pectin and substantial amounts of hemicellulose were solubilised. Anion exchange chromatography (AEC) showed co-elution of pectin and acetylated glucuronoxylan in tomato, representing 18% of solubilised uronic acid and 48% of solubilised xylose by ball milling from ChUS. The existence of a covalently linked pectin-xylan complex was proposed since xylan co-precipitated with pectin under mild alkali conditions. It was proposed that pectin links with xylan through the RG-I region since degradation of HG did not alter AEC elution patterns for RG-I and xylan, suggesting RG-I - xylan interactions.