Xylopentaose

Efficient production of xylooligosaccharides from Camellia oleifera shells pretreated by pyruvic acid at lower temperature.[Pubmed:38199559]

Int J Biol Macromol. 2024 Feb;259(Pt 2):129262.

XOS production from lignocellulose using organic carboxylic acids and alkyd acids has been widely reported. However, it still faces harsh challenges such as high energy consumption, high cost, and low purity. Pyruvic acid (PYA), a carbonyl acid with carbonyl and carboxyl groups, was used to produce XOS due to its stronger catalytic activity. In this work, XOS was efficiently prepared from COS in an autoclave under the condition of 0.21 M PYA-121 degrees C-35 min. The total yield of XOS reached 68.72 % without producing any toxic by-products, including furfural (FF) and 5-hydroxymethylfurfural (5-HMF). The yield of xylobiose (X(2)), xylotriose (X(3)), xylotetraose (X(4)), and Xylopentaose (X(5)) were 20.58 %, 12.47 %, 15.74 %, and 10.05 %, respectively. Meanwhile, 89.05 % of lignin was retained in the solid residue, which provides a crucial functional group for synthesizing layered carbon materials (SRG-a). It achieves excellent electromagnetic shielding (EMS) performance through graphitization, reaching -30 dB at a thickness of 2.0 mm. The use of a PYA catalyst in the production of XOS has proven to be an efficient method due to lower temperature, lower acid consumption, and straightforward operation.

In vitro evaluation of the application of an optimized xylanase cocktail for improved monogastric feed digestibility.[Pubmed:38169048]

J Anim Physiol Anim Nutr (Berl). 2024 Jan 3.

Xylanases from glycoside hydrolase (GH) families 10 and 11 are common feed additives for broiler chicken diets due to their catalytic activity on the nonstarch polysaccharide xylan. This study investigated the potential of an optimized binary GH10 and GH11 xylanase cocktail to mitigate the antinutritional effects of xylan on the digestibility of locally sourced chicken feed. Immunofluorescence visualization of the activity of the xylanase cocktail on xylan in the yellow corn of the feed showed a substantial collapse in the morphology of cell walls. Secondly, the reduction in the viscosity of the digesta of the feed by the cocktail showed an effective degradation of the soluble fraction of xylan. Analysis of the xylan degradation products from broiler feeds by the xylanase cocktail showed that xylotriose and Xylopentaose were the major xylooligosaccharides (XOS) produced. In vitro evaluation of the prebiotic potential of these XOS showed that they improved the growth of the beneficial bacteria Streptococcus thermophilus and Lactobacillus bulgaricus. The antibacterial activity of broths from XOS-supplemented probiotic cultures showed a suppressive effect on the growth of the extraintestinal infectious bacterium Klebsiella pneumoniae. Supplementing the xylanase cocktail in cereal animal feeds attenuated xylan's antinutritional effects by reducing digesta viscosity and releasing entrapped nutrients. Furthermore, the production of prebiotic XOS promoted the growth of beneficial bacteria while inhibiting the growth of pathogens. Based on these effects of the xylanase cocktail on the feed, improved growth performance and better feed conversion can potentially be achieved during poultry rearing.

Discovery of a bifunctional xylanolytic enzyme with arabinoxylan arabinofuranohydrolase-d3 and endo-xylanase activities and its application in the hydrolysis of cereal arabinoxylans.[Pubmed:37096984]

Microb Biotechnol. 2023 Jul;16(7):1536-1547.

Xylanolytic enzymes, with both endo-xylanase and arabinoxylan arabinofuranohydrolase (AXH) activities, are attractive for the economically feasible conversion of recalcitrant arabinoxylan. However, their characterization and utilization of these enzymes in biotechnological applications have been limited. Here, we characterize a novel bifunctional enzyme, rAbf43A, cloned from a bacterial consortium that exhibits AXH and endo-xylanase activities. Hydrolytic pattern analyses revealed that the AXH activity belongs to AXHd3 because it attacked only the C(O)-3-linked arabinofuranosyl residues of double-substituted xylopyranosyl units of arabinoxylan and arabinoxylan-derived oligosaccharides, which are usually resistant to hydrolysis. The enzyme rAbf43A also liberated a series of xylo-oligosaccharides (XOSs) from beechwood xylan, xylohexaose and Xylopentaose, indicating that rAbf43A exhibited endo-xylanase activity. Homology modelling based on AlphaFold2 and site-directed mutagenesis identified three non-catalytic residues (H161, A270 and L505) located in the substrate-binding pocket essential for its dual-functionality, while the mutation of A117 located in the -1 subsite to the proline residue only affected its endo-xylanase activity. Additionally, rAbf43A showed significant synergistic action with the bifunctional xylanase/feruloyl esterase rXyn10A/Fae1A from the same bacterial consortium on insoluble wheat arabinoxylan and de-starched wheat bran degradation. When rXyn10A/Fae1A was added to the rAbf43A pre-hydrolyzed reactions, the amount of released reducing sugars, xylose and ferulic acid increased by 9.43% and 25.16%, 189.37% and 93.54%, 31.39% and 32.30%, respectively, in comparison with the sum of hydrolysis products released by each enzyme alone. The unique characteristics of rAbf43A position it as a promising candidate not only for designing high-performance enzyme cocktails but also for investigating the structure-function relationship of GH43 multifunctional enzymes.

Xylanopectinolytic enzymes by marine actinomycetes from sediments of Sarena Kecil, North Sulawesi: high potential to produce galacturonic acid and xylooligosaccharides from raw biomass.[Pubmed:36920661]

J Genet Eng Biotechnol. 2023 Mar 15;21(1):31.

BACKGROUND: Actinomycetes isolated from marine habitats are known to have the potential for novel enzymes that are beneficial in the industry. In-depth knowledge is necessary given the variety of this bacterial group in Indonesia and the lack of published research. Actinomycetes isolates (BLH 5-14) obtained from marine sediments of Sarena Kecil, Bitung, North Sulawesi, Indonesia, showed an ability to produce pectinase and xylanase that have equal or even higher potential for pectic-oligosaccharides (POS) and xylooligosaccharides (XOS) production from raw biomass than from commercial substrates. This study's objective was to characterize both enzymes to learn more for future research and development. RESULTS: Pectinase had the highest activity on the 6(th) day (1.44+/-0.08 U/mL) at the optimum pH of 8.0 and optimum temperature of 50 degrees C. Xylanase had the maximum activity on the 6(th) day (4.33+/-0.03 U/mL) at optimum pH 6.0 and optimum temperature 60 degrees C. Hydrolysis and thin layer chromatography also showed that pectinase was able to produce monosaccharides such as galacturonic acid (P1), and xylanase was able to yield oligosaccharides such as xylotriose (X3), xylotetraose (X4), and Xylopentaose (X5). BLH 5-14 identified as the genus Streptomyces based on the 16S rDNA sequences and the closely related species Streptomyces tendae (99,78%). CONCLUSIONS: In the eco-friendly paper bleaching industry, Streptomyces tendae has demonstrated the potential to create enzymes with properties that can be active in a wide range of pH levels. The oligosaccharides have the potential as prebiotics or dietary supplements with anti-cancer properties. Further research is needed to optimize the production, purification, and development of the application of pectinase and xylanase enzymes produced by Actinomycetes isolates.

Substrate Specificities of GH8, GH39, and GH52 beta-xylosidases from Bacillus halodurans C-125 Toward Substituted Xylooligosaccharides.[Pubmed:33394289]

Appl Biochem Biotechnol. 2021 Apr;193(4):1042-1055.

Substrate specificities of glycoside hydrolase families 8 (Rex), 39 (BhXyl39), and 52 (BhXyl52) beta-xylosidases from Bacillus halodurans C-125 were investigated. BhXyl39 hydrolyzed xylotriose most efficiently among the linear xylooligosaccharides. The activity decreased in the order of xylohexaose > Xylopentaose > xylotetraose and it had little effect on xylobiose. In contrast, BhXyl52 hydrolyzed xylobiose and xylotriose most efficiently, and its activity decreased when the main chain became longer as follows: xylotetraose > Xylopentaose > xylohexaose. Rex produced O-beta-D-xylopyranosyl-(1 --> 4)-[O-alpha-L-arabinofuranosyl-(1 --> 3)]-O-beta-D-xylopyranosyl-(1 --> 4)-beta-D-xylopyranose (Ara(2)Xyl(3)) and O-beta-D-xylopyranosyl-(1 --> 4)-[O-4-O-methyl-alpha-D-glucuronopyranosyl-(l --> 2)]-beta-D-xylopyranosyl-(1 --> 4)-beta-D-xylopyranose (MeGlcA(2)Xyl(3)), which lost a xylose residue from the reducing end of O-beta-D-xylopyranosyl-(1 --> 4)-[O-alpha-L-arabinofuranosyl-(1 --> 3)]-O-beta-D-xylopyranosyl-(1 --> 4)-beta-D-xylopyranosyl-(1 --> 4)-beta-D-xylopyranose (Ara(3)Xyl(4)) and O-beta-D-xylopyranosyl-(1 --> 4)-[O-4-O-methyl-alpha-D-glucuronopyranosyl-(1 --> 2)]-beta-D-xylopyranosyl-(1 --> 4)-beta-D-xylopyranosyl-(1 --> 4)-beta-D-xylopyranose (MeGlcA(3)Xyl(4)). It was considered that there is no space to accommodate side chains at subsite -1. BhXyl39 rapidly hydrolyzes the non-reducing-end xylose linkages of MeGlcA(3)Xyl(4), while the arabinose branch does not significantly affect the enzyme activity because it degrades Ara(3)Xyl(4) as rapidly as unmodified xylotetraose. The model structure suggested that BhXyl39 enhanced the activity for MeGlcA(3)Xyl(4) by forming a hydrogen bond between glucuronic acid and Lys265. BhXyl52 did not hydrolyze Ara(3)Xyl(4) and MeGlcA(3)Xyl(4) because it has a narrow substrate binding pocket and 2- and 3-hydroxyl groups of xylose at subsite +1 hydrogen bond to the enzyme.

Coproduction of xylooligosaccharides and fermentable sugars from sugarcane bagasse by seawater hydrothermal pretreatment.[Pubmed:32325380]

Bioresour Technol. 2020 Aug;309:123385.

In this study, natural seawater without additional chemicals was selected to treat sugarcane bagasse for the production of xylooligosaccharides and glucose. This pretreatment not only more effectively conserves freshwater resources than hydrothermal pretreatment and enzymatic hydrolysis, but also decreases corrosion of the equipment relative to techniques utilizing acid and alkaline pretreatment. The maximum yield of 67.12% xylooligosaccharides (of initial xylan), including 11.49% xylobiose, 16.23% xylotriose, 23.82% xylotetraose, and 15.58% Xylopentaose was obtained under mild condition (175 degrees C for 30 min). Moreover, greater amounts of xylotetraose were generated during seawater hydrothermal pretreatment under all conditions, likely because NaCl in seawater cut the hydrogen bonds between xylo-oligomers. In addition, 94.69% cellulose digestibility and 78.58% xylan digestibility were achieved from the solid residue with an enzyme dosage of 30 FPU/g cellulose. Results indicated that seawater hydrothermal pretreatment is a more environmentally-friendly and sustainable technique for producing xylooligosaccharides and fermentable sugars than other methods.

An endoxylanase rapidly hydrolyzes xylan into major product xylobiose via transglycosylation of xylose to xylotriose or xylotetraose.[Pubmed:32241400]

Carbohydr Polym. 2020 Jun 1;237:116121.

Here, we proposed an effective strategy to enhance a novel endoxylanase (Taxy11) activity and elucidated an efficient catalysis mechanism to produce xylooligosaccharides (XOSs). Codon optimization and recruitment of natural propeptide in Pichia pastoris resulted in achievement of Taxy11 activity to 1405.65 +/- 51.24 U/mL. Analysis of action mode reveals that Taxy11 requires at least three xylose (xylotriose) residues for hydrolysis to yield xylobiose. Results of site-directed mutagenesis indicate that residues Glu(119), Glu(210), and Asp(53) of Taxy11 are key catalytic sites, while Asp(203) plays an auxiliary role. The novel mechanism whereby Taxy11 catalyzes conversion of xylan or XOSs into major product xylobiose involves transglycosylation of xylose to xylotriose or xylotetraose as substrate, to form xylotetraose or Xylopentaose intermediate, respectively. Taxy11 displayed highly hydrolytic activity toward corncob xylan, producing 50.44 % of xylobiose within 0.5 h. This work provides a cost-effective and sustainable way to produce value-added biomolecules XOSs (xylobiose-enriched) from agricultural waste.

Xylooligosaccharides chemical stability after high-intensity ultrasound processing of prebiotic orange juice.[Pubmed:31945564]

Ultrason Sonochem. 2020 May;63:104942.

The effects of the high-intensity ultrasound (HIUS) technology at the nominal powers of 300, 600, 900, and 1200 W were evaluated on the chemical stability of xylooligosaccharides (XOS) used to enrich orange juice. The ultrasound energy performance for each nominal power applied to the XOS-enriched orange juice was determined by calculating acoustic powers (W), HIUS intensity (W/cm(2)), and energy density (kJ/mL). Physicochemical properties (pH and soluble solid content), organic acid content (ascorbic, malic, and citric acids), total phenolic content (TPC), antioxidant activity by the FRAP (Ferric reducing ability of plasma) method, sugar (glucose, fructose, and sucrose), and XOS (xylobiose, xylotriose, xylotetraose, Xylopentaose, and xylohexaose) content were determined. The pH and soluble solid content did not change after all HIUS treatments. The HIUS process severity was monitored by quantifying ascorbic acid content after the treatments. A significant linear decrease in the ascorbic acid content was observed in prebiotic orange juice with the HIUS process intensification by increasing nominal power. The malic acid and citric acid contents had similar behavior according to the HIUS process intensification. The nominal power increase from 300 to 600 W increased the concentration of both organic acids, however, the intensification up to 1200 W reduced their concentration in the functional beverage. The TPC and FRAP data corroborated with the results observed for the ascorbic acid content. However, the HIUS processing did not alter sugar and XOS contents. The XOS chromatographic profiles were not modified by the HIUS treatment and presented the same amount of all prebiotic compounds before and after the HIUS treatment. Overall, HIUS technology has been evaluated as a promising stabilization technique for prebiotic beverages enriched with XOS due to their high chemical stability to this emerging technology under severe process conditions.

Laboratory Evolution of GH11 Endoxylanase Through DNA Shuffling: Effects of Distal Residue Substitution on Catalytic Activity and Active Site Architecture.[Pubmed:31824938]

Front Bioeng Biotechnol. 2019 Nov 22;7:350.

Endoxylanase with high specific activity, thermostability, and broad pH adaptability is in huge demand. The mutant library of GH11 endoxylanase was constructed via DNA shuffling by using the catalytic domain of Bacillus amyloliquefaciens xylanase A (BaxA) and Thermomonospora fusca TF xylanase A (TfxA) as parents. A total of 2,250 colonies were collected and 756 of them were sequenced. Three novel mutants (DS153: N29S, DS241: S31R and DS428: I51V) were identified and characterized in detail. For these mutants, three residues of BaxA were substituted by the corresponding one of TfxA_CD. The specific activity of DS153, DS241, and DS428 in the optimal condition was 4.54, 4.35, and 3.9 times compared with the recombinant BaxA (reBaxA), respectively. The optimum temperature of the three mutants was 50 degrees C. The optimum pH for DS153, DS241, and DS428 was 6.0, 7.0, and 6.0, respectively. The catalytic efficiency of DS153, DS241, and DS428 enhanced as well, while their sensitivity to recombinant rice xylanase inhibitor (RIXI) was lower than that of reBaxA. Three mutants have identical hydrolytic function as reBaxA, which released xylobiose-Xylopentaose from oat spelt, birchwood, and beechwood xylan. Furthermore, molecular dynamics simulations were performed on BaxA and three mutants to explore the precise impact of gain-of-function on xylanase activity. The tertiary structure of BaxA was not altered under the substitution of distal residues (N29S, S31R, and I51V); it induced slightly changes in active site architecture. The distal impact rescued the BaxA from native conformation ("closed state") through weakening interactions between "gate" residues (R112, N35 in DS241 and DS428; W9, P116 in DS153) and active site residues (E78, E172, Y69, and Y80), favoring conformations with an "open state" and providing improved activity. The current findings would provide a better and more in-depth understanding of how distal single residue substitution improved the catalytic activity of xylanase at the atomic level.

Heterologous expression, purification and biochemical characterization of a new endo-1,4-beta-xylanase from Rhodothermaceae bacterium RA.[Pubmed:31376486]

Protein Expr Purif. 2019 Dec;164:105464.

Xylanases (EC 3.2.1.8) are essential enzymes due to their applications in various industries such as textile, animal feed, paper and pulp, and biofuel industries. Halo-thermophilic Rhodothermaceae bacterium RA was previously isolated from a hot spring in Malaysia. Genomic analysis revealed that this bacterium is likely to be a new genus of the family Rhodothermaceae. In this study, a xylanase gene (1140 bp) that encoded 379 amino acids from the bacterium was cloned and expressed in Escherichia coli BL21(DE3). Based on InterProScan, this enzyme XynRA1 contained a GH10 domain and a signal peptide sequence. XynRA1 shared low similarity with the currently known xylanases (the closest is 57.2-65.4% to Gemmatimonadetes spp.). The purified XynRA1 achieved maximum activity at pH 8 and 60  degrees C. The protein molecular weight was 43.1 kDa XynRA1 exhibited an activity half-life (t(1/2)) of 1 h at 60  degrees C and remained stable at 50  degrees C throughout the experiment. However, it was NaCl intolerant, and various types of salt reduced the activity. This enzyme effectively hydrolyzed xylan (beechwood, oat spelt, and Palmaria palmata) and xylodextrin (xylotriose, xylotetraose, Xylopentaose, and xylohexaose) to produce predominantly xylobiose. This xylanase is the first functionally characterized enzyme from the bacterium, and this work broadens the knowledge of GH10 xylanases.

Sugar Composition in Asparagus Spears and Its Relationship to Soil Chemical Properties.[Pubmed:34354519]

J Appl Glycosci (1999). 2019 Feb 20;66(1):47-50.

Glycoside hydrolases require carboxyl groups as catalysts for their activity. A retaining xylanase from Streptomyces olivaceoviridis E-86 belonging to glycoside hydrolase family 10 possesses Glu128 and Glu236 that respectively function as acid/base and nucleophile. We previously developed a unique mutant of the retaining xylanase, N127S/E128H, whose deglycosylation is triggered by azide. A crystallographic study reported that the transient formation of a Ser-His catalytic dyad in the reaction cycle possibly reduced the azidolysis reaction. In the present study, we engineered a catalytic dyad with enhanced stability by site-directed mutagenesis and crystallographic study of N127S/E128H. Comparison of the Michaelis complexes of N127S/E128H with pNP-X(2) and with Xylopentaose showed that Ser127 could form an alternative hydrogen bond with Thr82, which disrupts the formation of the Ser-His catalytic dyad. The introduction of T82A mutation in N127S/E128H produces an enhanced first-order rate constant (6 times that of N127S/E128H). We confirmed the presence of a stable Ser-His hydrogen bond in the Michaelis complex of the triple mutant, which forms the productive tautomer of His128 that acts as an acid catalyst. Because the glycosyl azide is applicable in the bioconjugation of glycans by using click chemistry, the enzyme-assisted production of the glycosyl azide may contribute to the field of glycobiology.

Characterization of a novel xylanase from Aspergillus flavus with the unique properties in production of xylooligosaccharides.[Pubmed:30747436]

J Basic Microbiol. 2019 Apr;59(4):351-358.

A novel xylanase from the filamentous fungus Aspergillus flavus was purified and characterized as the beta-1, 4-endoxylanase (designed as AfXynB) with a molecular mass (32.2 kDa), which is different from all of the previously reported xylanases from the same strain. AfXynB was optimally active at pH 7.5 and 55 degrees C, respectively. It was stable up to 50 degrees C within range of pH 4.0-9.5, and displayed an excellent tolerance to various cations, reagents, and proteases. AfXynB showed specific activity toward beechwood xylan but no detected activity toward CMC and pNP-beta-D-xylopyranoside. The xylanase is a typical endo-xylanase; it could hydrolyze beechwood xylan to only yield xylobiose (X2) and Xylopentaose (X5). Actually, this may be the first report for the endo-xylanases that displayed such a unique hydrolytic property. These findings in the present study have great implications for its future applications of the novel xylanase.

Discovery of a Thermostable GH10 Xylanase with Broad Substrate Specificity from the Arctic Mid-Ocean Ridge Vent System.[Pubmed:30635385]

Appl Environ Microbiol. 2019 Mar 6;85(6):e02970-18.

A two-domain GH10 xylanase-encoding gene (amor_gh10a) was discovered from a metagenomic data set, generated after in situ incubation of a lignocellulosic substrate in hot sediments on the sea floor of the Arctic Mid-Ocean Ridge (AMOR). AMOR_GH10A comprises a signal peptide, a carbohydrate-binding module belonging to a previously uncharacterized family, and a catalytic glycosyl hydrolase (GH10) domain. The enzyme shares the highest sequence identity (42%) with a hypothetical protein from a Verrucomicrobia bacterium, and its GH10 domain shares low identity (24 to 28%) with functionally characterized xylanases. Purified AMOR_GH10A showed thermophilic and halophilic properties and was active toward various xylans. Uniquely, the enzyme showed high activity toward amorphous cellulose, glucomannan, and xyloglucan and was more active toward cellopentaose than toward Xylopentaose. Binding assays showed that the N-terminal domain of this broad-specificity GH10 binds strongly to amorphous cellulose, as well as to microcrystalline cellulose, birchwood glucuronoxylan, barley beta-glucan, and konjac glucomannan, confirming its classification as a novel CBM (CBM85).IMPORTANCE Hot springs at the sea bottom harbor unique biodiversity and are a promising source of enzymes with interesting properties. We describe the functional characterization of a thermophilic and halophilic multidomain xylanase originating from the Arctic Mid-Ocean Ridge vent system, belonging to the well-studied family 10 of glycosyl hydrolases (GH10). This xylanase, AMOR_GH10A, has a surprisingly wide substrate range and is more active toward cellopentaose than toward Xylopentaose. This substrate promiscuity is unique for the GH10 family and could prove useful in industrial applications. Emphasizing the versatility of AMOR_GH10A, its N-terminal domain binds to both xylans and glycans, while not showing significant sequence similarities to any known carbohydrate-binding module (CBM) in the CAZy database. Thus, this N-terminal domain lays the foundation for the new CBM85 family.

In vitro fermentation of O‑acetyl‑arabinoxylan from bamboo shavings by human colonic microbiota.[Pubmed:30521907]

Int J Biol Macromol. 2019 Mar 15;125:27-34.

BSH-1 is an O‑acetyl-arabinoxylan obtained from bamboo shavings. This study investigated its fermentation behavior by human colonic microbiota in vitro. Results showed that BSH-1 remarkably modulated the composition of human colonic microbiota, mainly by increasing the growth of potential beneficial genera (i.e. Bifidobacterium, Lactobacillus, Bacteroides, Prevotella_7, Parabacteroides) and by decreasing the growth of potential harmful genera (i.e. Fusobacterium, Lachnospiraceae_UCG-008, Bilophila and Desulfovibrio). BSH-1 significantly promoted the production of short-chain fatty acids, especially acetic, propionic and n-butyric acids. After 48 h fermentation, the concentration of n-butyric acid in BSH-1 fermentation culture was increased by 2.41 times compared to the blank. During fermentation, the activity of acetyl xylan esterase, arabinofuranosidase, xylanase and xylosidase was enhanced. Moreover, free arabinose, xylose, xylobiose, xylotriose, xylotetraose, Xylopentaose and xylohexaose were detected. These results suggest that BSH-1 could potentially be a functional ingredient to improve gut health.