RESUMO
Herbivorax saccincola A7 is an anaerobic alkali-thermophilic lignocellulolytic bacterium that possesses a cellulosome and high xylan degradation ability. To understand the expression profile of extracellular enzymes by carbon sources, quantitative real-time PCR was performed on all cellulosomal and non-cellulosomal enzyme genes of H. saccincola A7 using cellulose and xylan as carbon sources. The results confirmed that the scaffolding proteins of H. saccincola A7 were expressed. In general, the cellulosomal genes belonging to the glycoside hydrolase families 9, 10, 11, and 48 were repressed when xylan was the sole carbon source, but these genes were significantly induced in the presence of cellulose. These results indicate that cellulose, not xylan, is a key inducer of cellulosomal genes in H. saccincola A7. The RsgI-like proteins, which regulate a carbohydrate-sensing mechanism in Clostridium thermocellum, were also found to be encoded in the H. saccincola A7 genome. To confirm the regulation by RsgI-like proteins, the relative expression of σI1-σI4 factors was analyzed on both carbon sources. The expression of alternative σI1 and σI2 factors was enhanced by the presence of cellulose. By contrast, the expression of σI3 and σI4 factors was activated by both cellulose and xylan. Taken together, the results reveal that the cellulosomal and non-cellulosomal genes of H. saccincola A7 are regulated through a carbohydrate-sensing mechanism involving anti-σ regulator RsgI-like proteins. KEY POINTS: ⢠qRT-PCR performed on cellulosomal and non-cellulosomal genes of H. saccincola A7 ⢠Cellulose is a key inducer of the cellulosome of H. saccincola A7 ⢠H. saccincola A7 possesses a similar system of anti-σ regulator RsgI-like proteins.
Assuntos
Celulose , Celulossomas , Regulação Bacteriana da Expressão Gênica , Xilanos , Celulossomas/metabolismo , Celulossomas/genética , Celulose/metabolismo , Xilanos/metabolismo , Polissacarídeos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Australian saltbush (Atriplex spp.) survive in exceptionally saline environments and are often used for pasture in semi-arid areas. To investigate the impact of salinity on saltbush root morphology and root exudates, three Australian native saltbush species (Atriplex nummularia , Atriplex amnicola , and Atriplex vesicaria ) were grown in vitro in optimised sterile, semi-hydroponic systems in media supplemented with different concentrations of salt (NaCl). Histological stains and chromatographic techniques were used to characterise the root apical meristem (RAM) type and root exudate composition of the saltbush seedlings. We report that saltbush species have closed-type RAMs, which release border-like cells (BLCs). Monosaccharide content, including glucose and fructose, in the root mucilage of saltbush was found to be uniquely low, suggesting that saltbush may minimise carbon release in polysaccharides of root exudates. Root mucilage also contained notable levels of salt, plus increasing levels of unidentified compounds at peak salinity. Un-esterified homogalacturonan, xyloglucan, and arabinogalactan proteins between and on the surface of BLCs may aid intercellular adhesion. At the highest salinity levels, root cap morphology was altered but root:shoot ratio remained consistent. While questions remain about the identity of some components in saltbush root mucilage other than the key monosaccharides, this new information about root cap morphology and cell surface polysaccharides provides avenues for future research.
Assuntos
Atriplex , Meristema , Raízes de Plantas , Plântula , Plântula/efeitos dos fármacos , Plântula/metabolismo , Plântula/crescimento & desenvolvimento , Meristema/efeitos dos fármacos , Meristema/citologia , Meristema/metabolismo , Raízes de Plantas/metabolismo , Raízes de Plantas/efeitos dos fármacos , Atriplex/efeitos dos fármacos , Atriplex/metabolismo , Cloreto de Sódio/farmacologia , Mucilagem Vegetal/metabolismo , Xilanos/metabolismo , Glucanos/metabolismo , SalinidadeRESUMO
This study identified a salt-tolerant GH11 xylanase, Xynst, which was isolated from a soil bacterium Bacillus sp. SC1 and can resist as high as 4 M NaCl. After rational design and high-throughput screening of site-directed mutant libraries, a double mutant W6F/Q7H with a 244% increase in catalytic activity and a 10 °C increment in optimal temperature was obtained. Both Xynst and W6F/Q7H xylanases were stimulated by high concentrations of salts. In particular, the activity of W6F/Q7H was more than eight times that of Xynst in the presence of 2 M NaCl at 65 °C. Kinetic parameters indicated they have the highest affinity for beechwood xylan (Km = 0.30 mg mL-1 for Xynst and 0.18 mg mL-1 for W6F/Q7H), and W6F/Q7H has very high catalytic efficiency (Kcat/Km = 15483.33 mL mg-1 s-1). Molecular dynamic simulation suggested that W6F/Q7H has a more compact overall structure, improved rigidity of the active pocket edge, and a flexible upper-end alpha helix. Hydrolysis of different xylans by W6F/Q7H released more xylooligosaccharides and yielded higher proportions of xylobiose and xylotriose than Xynst did. The conversion efficiencies of Xynst and W6F/Q7H on all tested xylans exceeded 20%, suggesting potential applications in the agricultural and food industries.
Assuntos
Bacillus , Endo-1,4-beta-Xilanases , Glucuronatos , Oligossacarídeos , Engenharia de Proteínas , Oligossacarídeos/metabolismo , Oligossacarídeos/química , Glucuronatos/metabolismo , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Endo-1,4-beta-Xilanases/química , Bacillus/enzimologia , Bacillus/genética , Engenharia de Proteínas/métodos , Simulação de Dinâmica Molecular , Cloreto de Sódio/farmacologia , Cinética , Xilanos/metabolismo , Mutagênese Sítio-Dirigida , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Hidrólise , DissacarídeosRESUMO
Cotton is the most common natural fibre used in textile manufacture, used alone or with other fibres to create a wide range of fashion clothing and household textiles. Most of these textiles are cleaned using detergents and domestic or commercial washing machines using processes that require many chemicals and large quantities of water and energy. Enzymes can reduce this environmental footprint by enabling effective detergency at reduced temperatures, mostly by directly attacking substrates present in the soils. In the present study, we report the contribution of a cleaning cellulase enzyme based on the family 44 glycoside hydrolase (GH) endo-beta-1,4-glucanase from Paenibacillus polymyxa. The action of this enzyme on textile fibres improves laundry detergent performance in several vectors including soil anti-redeposition, dye transfer inhibition and stain removal. Molecular probes are used to study how this enzyme is targeting both amorphous cellulose and xyloglucan on textile fibres and the relationship between textile surface effects and observed performance benefits.
Assuntos
Fibra de Algodão , Detergentes , Detergentes/química , Paenibacillus/enzimologia , Têxteis , Polissacarídeos/química , Polissacarídeos/metabolismo , Celulase/metabolismo , Celulase/química , Celulose/química , Celulose/metabolismo , Xilanos/química , Xilanos/metabolismo , Glucanos/química , Glucanos/metabolismoRESUMO
Paddy fields are a major emission source of the greenhouse gas methane. In the present study, the addition of ferrihydrite to xylan-amended paddy soil microcosms suppressed methane emissions. PCR-based and metatranscriptomic ana-lyses revealed that the addition of ferrihydrite suppressed methanogenesis by heterogeneous methanogens and simultaneously activated Geobacteraceae, the most abundant iron-reducing diazotrophs. Geobacteraceae may preferentially metabolize xylan and/or xylan-derived carbon compounds that are utilized by methanogens. Geomonas terrae R111 utilized xylan as a growth substrate under liquid culture conditions. This may constitute a novel mechanism for the mitigation of methane emissions previously observed in ferric iron oxide-applied paddy field soils.
Assuntos
Compostos Férricos , Metano , Microbiologia do Solo , Xilanos , Metano/metabolismo , Compostos Férricos/metabolismo , Xilanos/metabolismo , Solo/química , Oxirredução , Ferro/metabolismoRESUMO
The primary plant cell wall (PCW) is a specialized structure composed predominantly of cellulose, hemicelluloses and pectin. While the role of cellulose and hemicelluloses in the formation of the PCW scaffold is undeniable, the mechanisms of how hemicelluloses determine the mechanical properties of PCW remain debatable. Thus, we produced bacterial cellulose-hemicellulose hydrogels as PCW analogues, incorporated with hemicelluloses. Next, we treated samples with hemicellulose degrading enzymes, and explored its structural and mechanical properties. As suggested, difference of hemicelluloses in structure and chemical composition resulted in a variety of the properties studied. By analyzing all the direct and indirect evidences we have found that glucomannan, xyloglucan and arabinoxylan increased the width of cellulose fibers both by hemicellulose surface deposition and fiber entrapment. Arabinoxylan increased stresses and moduli of the hydrogel by its reinforcing effect, while for xylan, increase in mechanical properties was determined by establishment of stiff cellulose-cellulose junctions. In contrast, increasing content of xyloglucan decreased stresses and moduli of hydrogel by its weak interactions with cellulose, while glucomannan altered cellulose network formation via surface deposition, decreasing its strength. The current results provide evidence for structure-dependent mechanisms of cellulose-hemicellulose interactions, suggesting the specific structural role of the latter.
Assuntos
Celulose , Glucanos , Hidrogéis , Mananas , Polissacarídeos , Xilanos , Hidrogéis/química , Polissacarídeos/química , Celulose/química , Xilanos/química , Xilanos/metabolismo , Mananas/química , Glucanos/química , Glucanos/biossíntese , Glucanos/metabolismo , Parede Celular/metabolismo , Parede Celular/químicaRESUMO
A transformation in plant cell wall evolution marked the emergence of grasses, grains and related species that now cover much of the globe. Their tough, less digestible cell walls arose from a new pattern of cross-linking between arabinoxylan polymers with distinctive ferulic acid residues. Despite extensive study, the biochemical mechanism of ferulic acid incorporation into cell walls remains unknown. Here we show that ferulic acid is transferred to arabinoxylans via an unexpected sucrose derivative, 3,6-O-diferuloyl sucrose (2-feruloyl-O-α-D-glucopyranosyl-(1'â2)-3,6-O-feruloyl-ß-D-fructofuranoside), formed by a sucrose ferulate cycle. Sucrose gains ferulate units through sequential transfers from feruloyl-CoA, initially at the O-3 position of sucrose catalysed by a family of BAHD-type sucrose ferulic acid transferases (SFT1 to SFT4 in maize), then at the O-6 position by a feruloyl sucrose feruloyl transferase (FSFT), which creates 3,6-O-diferuloyl sucrose. An FSFT-deficient mutant of maize, disorganized wall 1 (dow1), sharply decreases cell wall arabinoxylan ferulic acid content, causes accumulation of 3-O-feruloyl sucrose (α-D-glucopyranosyl-(1'â2)-3-O-feruloyl-ß-D-fructofuranoside) and leads to the abortion of embryos with defective cell walls. In vivo, isotope-labelled ferulic acid residues are transferred from 3,6-O-diferuloyl sucrose onto cell wall arabinoxylans. This previously unrecognized sucrose ferulate cycle resolves a long-standing mystery surrounding the evolution of the distinctive cell wall characteristics of cereal grains, biofuel crops and related commelinid species; identifies an unexpected role for sucrose as a ferulate group carrier in cell wall biosynthesis; and reveals a new paradigm for modifying cell wall polymers through ferulic acid incorporation.
Assuntos
Parede Celular , Ácidos Cumáricos , Sacarose , Xilanos , Ácidos Cumáricos/metabolismo , Xilanos/metabolismo , Sacarose/metabolismo , Parede Celular/metabolismo , Parede Celular/química , Zea mays/metabolismo , Zea mays/genéticaRESUMO
This research investigates the influence of milling methods and starter cultures on the structural characteristics of water-extractable arabinoxylans (WE-AX) in stone-milled whole-grain flour and sourdough bread. Stone milling was conducted to generate six different whole wheat flour samples, from which sourdough bread was produced using wheat and rye starter cultures. The study found that both milling parameters and the type of starter culture significantly impacted the fine structural details of WE-AX, including sugar composition, arabinoxylan (AX) content, and the arabinose-to-xylose (A/X) ratio values. These differences were statistically significant (p < 0.05). Furthermore, transforming flour into sourdough bread resulted in the molecular degradation of AX, significantly reducing its molecular weight and leading to a more heterogeneous fine structure. This detailed characterization of AX's alterations during food processing provides insights into evaluating its potential health benefits in whole-grain products.
Assuntos
Pão , Farinha , Xilanos , Pão/análise , Triticum/química , Fermentação , Farinha/análise , Xilanos/análise , Xilanos/metabolismo , Ressonância Magnética Nuclear BiomolecularRESUMO
Hemicellulose is key in determining the fate of plant cell wall in almost all growth and developmental stages. Nevertheless, there is limited knowledge regarding its involvement in the development and ripening of banana fruit. This study investigated changes in the temporal-spatial distribution of various hemicellulose components, hemicellulose content, activities of the main hydrolysis enzymes, and transcription level of the main hemicellulose-related gene families in banana peels. Both hemicellulose and xylan contents were positively correlated to the fruit firmness observed in our previous study. On the contrary, the xylanase activity was negatively correlated to xylan content and the fruit firmness. The vascular bundle cells, phloem, and cortex of bananas are abundant in xyloglucan, xylan, and mannan contents. Interestingly, the changes in the signal intensity of the CCRC-M104 antibody recognizing non-XXXG type xyloglucan are positively correlated to hemicellulose content. According to RNA-Seq analysis, xyloglucan and xylan-related genes were highly active in the early stages of growth, and the expression of MaMANs and MaXYNs increased as the fruit ripened. The abundance of plant hormonal and growth-responsive cis-acting elements was detected in the 2 kb upstream region of hemicellulose-related gene families. Interaction between hemicellulose and cell wall-specific proteins and MaKCBP1/2, MaCKG1, and MaHKL1 was found. The findings shed light on cell wall hemicellulose's role in banana fruit development and ripening, which could improve nutrition, flavor, and reduce postharvest fruit losses.
Assuntos
Frutas , Musa , Polissacarídeos , Musa/metabolismo , Musa/genética , Musa/crescimento & desenvolvimento , Polissacarídeos/metabolismo , Frutas/metabolismo , Frutas/crescimento & desenvolvimento , Frutas/genética , Xilanos/metabolismo , Regulação da Expressão Gênica de Plantas , Glucanos/metabolismo , Parede Celular/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genéticaRESUMO
Exploring nutritional therapies that manipulate tryptophan metabolism to activate AhR signaling represents a promising approach for mitigating chronic colitis. Arabinoxylan is a bioactive constituent abundant in wheat bran. Here, we comprehensively investigated anti-colitis potentials of wheat bran arabinoxylan (WBAX), its synbiotic and postbiotic derived from WBAX and Limosilactobacillus reuteri WX-94 (i.e., a probiotic strain exhibiting tryptophan metabolic activity). WBAX fueled L. reuteri and promoted microbial conversion of tryptophan to AhR ligands during in vitro fermentation in the culture medium and in the fecal microbiota from type 2 diabetes. The WBAX postbiotic outperformed WBAX and its synbiotic in augmenting efficacy of tryptophan in restoring DSS-disturbed serum immune markers, colonic tight junction proteins and gene profiles involved in amino acid metabolism and FoxO signaling. The WBAX postbiotic remodeled gut microbiota and superiorly enhanced AhR ligands (i.e., indole metabolites and bile acids), alongside with elevation in colonic AhR and IL-22. Associations between genera and metabolites modified by the postbiotic and colitis in human were verified and strong binding capacities between metabolites and colitis-related targets were demonstrated by molecular docking. Our study advances the novel perspective of WBAX in manipulating tryptophan metabolism and anti-colitis potentials of WBAX postbiotic via promoting gut microbiota-dependent AhR signaling.
Assuntos
Colite , Fibras na Dieta , Microbioma Gastrointestinal , Limosilactobacillus reuteri , Simbióticos , Xilanos , Xilanos/farmacologia , Xilanos/química , Xilanos/metabolismo , Limosilactobacillus reuteri/metabolismo , Colite/metabolismo , Colite/microbiologia , Microbioma Gastrointestinal/efeitos dos fármacos , Fibras na Dieta/metabolismo , Humanos , Animais , Camundongos , Receptores de Hidrocarboneto Arílico/metabolismo , Triptofano/metabolismo , Simulação de Acoplamento Molecular , Fermentação , Masculino , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/microbiologia , MultiômicaRESUMO
A gene encoding a polysaccharide-degrading enzyme was cloned from the genome of the bacterium Nocardiopsis halotolerans. Analysis of the amino acid sequence of the protein showed the presence of the catalytic domain of the endo-1,4-ß-xylanases of the GH11 family. The gene was amplified by PCR and ligated into the pPic9m vector. A recombinant producer based on Pichia pastoria was obtained. The production of the enzyme, which we called NhX1, was carried out in a 10 L fermenter. Enzyme production was 10.4 g/L with an activity of 927 U/mL. Purification of NhX1 was carried out using Ni-NTA affinity chromatography. The purified enzyme catalyzed the hydrolysis of xylan but not other polysaccharides. Endo-1,4-ß-xylanase NhX1 showed maximum activity and stability at pH 6.0-7.0. The enzyme showed high thermal stability, remaining active at 90 °C for 20 min. With beechwood xylan, the enzyme showed Km 2.16 mg/mL and Vmax 96.3 U/mg. The products of xylan hydrolysis under the action of NhX1 were xylobiose, xylotriose, xylopentaose, and xylohexaose. Endo-1,4-ß-xylanase NhX1 effectively saccharified xylan-containing products used for the production of animal feed. The xylanase described herein is a thermostable enzyme with biotechnological potential produced in large quantities by P. pastoria.
Assuntos
Endo-1,4-beta-Xilanases , Estabilidade Enzimática , Xilanos , Xilanos/metabolismo , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Endo-1,4-beta-Xilanases/química , Hidrólise , Actinobacteria/enzimologia , Actinobacteria/genética , Concentração de Íons de Hidrogênio , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Clonagem Molecular/métodos , Especificidade por Substrato , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Pichia/genética , Pichia/metabolismo , Actinomycetales/enzimologia , Actinomycetales/genética , Sequência de Aminoácidos , SaccharomycetalesRESUMO
Xylan is one of the major hemicelluloses in plant cell walls and its xylosyl backbone is often decorated at O-2 with glucuronic acid (GlcA) and/or methylglucuronic acid (MeGlcA) residues. The GlcA/MeGlcA side chains may be further substituted with 2-O-arabinopyranose (Arap) or 2-O-galactopyranose (Gal) residues in some plant species, but the enzymes responsible for these substitutions remain unknown. During our endeavor to investigate the enzymatic activities of Arabidopsis MUR3-clade members of the GT47 glycosyltransferase family, we found that one of them was able to transfer Arap from UDP-Arap onto O-2 of GlcA side chains of xylan, and thus it was named xylan 2-O-arabinopyranosyltransferase 1 (AtXAPT1). The function of AtXAPT1 was verified in planta by its T-DNA knockout mutation showing a loss of the Arap substitution on xylan GlcA side chains. Further biochemical characterization of XAPT close homologs from other plant species demonstrated that while the poplar ones had the same catalytic activity as AtXAPT1, those from Eucalyptus, lemon-scented gum, sea apple, 'Ohi'a lehua, duckweed and purple yam were capable of catalyzing both 2-O-Arap and 2-O-Gal substitutions of xylan GlcA side chains albeit with differential activities. Sequential reactions with XAPTs and glucuronoxylan methyltransferase 3 (GXM3) showed that XAPTs acted poorly on MeGlcA side chains, whereas GXM3 could efficiently methylate arabinosylated or galactosylated GlcA side chains of xylan. Furthermore, molecular docking and site-directed mutagenesis analyses of Eucalyptus XAPT1 revealed critical roles of several amino acid residues at the putative active site in its activity. Together, these findings establish that XAPTs residing in the MUR3 clade of family GT47 are responsible for 2-O-arabinopyranosylation and 2-O-galactosylation of GlcA side chains of xylan.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Glicosiltransferases , Xilanos , Xilanos/metabolismo , Arabidopsis/genética , Arabidopsis/enzimologia , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Glicosiltransferases/química , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/química , Parede Celular/metabolismo , Parede Celular/enzimologia , Arabinose/metabolismoRESUMO
Efficiently utilization of plant resources is heavily restricted by the resistance of lignocellulose in plant cells, which is related to the interlinkages of lignocellulose components. Hemicellulose in plant cell wall is bound to cellulose by hydrogen bond and linked with lignin in lignin-carbohydrate complex (LCC). In the xylan chain of hemicellulose, glucuronic acid (GA) is a typical side-group, which provides clues for us to label and locate hemicellulose. The way to label GA on the surface of pulp fibers obtained from pulping process is benefit to explore the deconstruction of lignocellulose. Herein, a new visualization method, fluorescence modified molecularly imprinted polymers (MIP) were applied to recognize and locate GA on the pulp fiber surface. The method combining fluorescence imaging and integrated 3D fiber structure verified the feasibility of the MIP for specific GA recognition. The results showed that xylan (represented by GA) was closely attached to lignin, distributed along the inner wall of pulp fiber cells, and gradually taken off from the inside edge of fiber cells with the deconstruction of lignocellulose. This research provided a basis to develop visualization bioimaging technology to identify biomass components.
Assuntos
Lignina , Polímeros Molecularmente Impressos , Xilanos , Xilanos/química , Xilanos/metabolismo , Polímeros Molecularmente Impressos/química , Lignina/química , Polissacarídeos/química , Polissacarídeos/metabolismo , Impressão Molecular/métodosRESUMO
Crop straws provide enormous lignocellulose resources transformable for sustainable biofuels and valuable bioproducts. However, lignocellulose recalcitrance basically restricts essential biomass enzymatic saccharification at large scale. In this study, the mushroom-derived cellobiohydrolase (LeGH7) was introduced into Trichoderma reesei (Rut-C30) to generate two desirable strains, namely GH7-5 and GH7-6. Compared to the Rut-C30 strain, both engineered strains exhibited significantly enhanced enzymatic activities, with ß-glucosidases, endocellulases, cellobiohydrolases, and xylanase activities increasing by 113 %, 140 %, 241 %, and 196 %, respectively. By performing steam explosion and mild alkali pretreatments with mature straws of five bioenergy crops, diverse lignocellulose substrates were effectively digested by the crude enzymes secreted from the engineered strains, leading to the high-yield hexoses released for bioethanol production. Notably, the LeGH7 enzyme purified from engineered strain enabled to act as multiple cellulases and xylanase at higher activities, interpreting how synergistic enhancement of enzymatic saccharification was achieved for distinct lignocellulose substrates in major bioenergy crops. Therefore, this study has identified a novel enzyme that is active for simultaneous hydrolyses of cellulose and xylan, providing an applicable strategy for high biomass enzymatic saccharification and bioethanol conversion in bioenergy crops.
Assuntos
Biocombustíveis , Biomassa , Celulose , Etanol , Xilanos , Xilanos/metabolismo , Celulose/metabolismo , Etanol/metabolismo , Hypocreales/enzimologia , Hypocreales/genética , Hypocreales/metabolismo , Lignina/metabolismo , Hidrólise , Celulose 1,4-beta-Celobiosidase/metabolismo , Celulose 1,4-beta-Celobiosidase/genéticaRESUMO
Cellulose and hemicellulose are the major structural ß-glycan polysaccharides in cell walls of land plants. They are characterized by a backbone of ß-(1,3)- and/or ß-(1,4)-linked sugars such as glucose, mannose, or xylose. The backbones of these polymers are produced by processive glycosyltransferases (GTs) called synthases having multiple transmembrane domains anchoring them to the membrane. Thus, they are among the most difficult membrane proteins to test in vitro and to purify. Recently, we developed an in vitro GT-array (i-GTray) platform and showed that non-processive type II membrane GTs could be produced via cell-free system in a soluble and active form and tested in this platform. To determine whether i-GT-ray platform is adequate for the production and testing of ß-glycan synthases, we tested five synthases involved in cellulose, xyloglucan, (gluco)mannan, and ß-(1,3)(1,4)-mixed-linkage glucan synthesis. Our results revealed unsuspected features of these enzymes. For example, all these synthases could be produced in a soluble and active form and are active in the absence of detergent or membrane lipids, and none of them required a primer for initiation of synthesis. All synthases produced ethanol-insoluble products that were susceptible to the appropriate hydrolases (i.e., cellulase, lichenase, mannanase). Using this platform, we showed that AtCslC4 and AtXXT1 interact directly to form an active xyloglucan synthase that produced xylosylated cello-oligosaccharides (up to three xylosyl residues) when supplied with UDP-Glc and UDP-Xyl. i-GTray platform represents a simple and powerful functional genomics tool for discovery of new insights of synthase activities and can be adapted to other enzymes.
Assuntos
Glicosiltransferases , Polissacarídeos , Glicosiltransferases/metabolismo , Polissacarídeos/metabolismo , Xilanos/metabolismo , Celulose/metabolismo , Glucanos/metabolismo , Arabidopsis/enzimologia , Arabidopsis/metabolismoRESUMO
Xylan, one of the most important structures and polysaccharides, plays critical roles in plant development, growth, and defense responses to pathogens. Glucuronic acid substitution of xylan (GUX) functions in xylan sidechain decoration, which is involved in a wide range of physiological processes in plants. However, the specifics of GUXs in trees remain unclear. In this study, the characterization and evolution of the GUX family genes in E. grandis, a fast-growing forest tree belonging to the Myrtaceae family, were performed. A total of 23 EgGUXs were identified from the E. grandis genome, of which all members contained motif 2, 3, 5, and 7. All GUX genes were phylogeneticly clustered into five distinct groups. Among them, EgGUX01~EgGUX05 genes were clustered into group III and IV, which were more closely related to the AtGUX1, AtGUX2, and AtGUX4 members of Arabidopsis thaliana known to possess glucuronyltransferase activity, while most other members were clustered into group I. The light-responsive elements, hormone-responsive elements, growth and development-responsive elements, and stress-responsive elements were found in the promoter cis-acting elements, suggesting the expression of GUX might also be regulated by abiotic factors. RNA-Seq data confirmed that EgGUX02, EgGUX03, and EgGUX10 are highly expressed in xylem, and EgGUX09, EgGUX10, and EgGUX14 were obviously responses to abiotic stresses. The results of this paper will provide a comprehensive determination of the functions of the EgGUX family members, which will further contribute to understanding E. grandis xylan formation.
Assuntos
Eucalyptus , Regulação da Expressão Gênica de Plantas , Família Multigênica , Filogenia , Xilanos , Eucalyptus/genética , Xilanos/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Genoma de Planta , Regiões Promotoras GenéticasRESUMO
Xylan is a fundamental structural polysaccharide in plant secondary cell walls and a valuable resource for biorefinery applications. Deciphering the molecular motifs of xylans that mediate their interaction with cellulose and lignin is fundamental to understand the structural integrity of plant cell walls and to design lignocellulosic materials. In the present study, we investigated the pattern of acetylation and glucuronidation substitution in hardwood glucuronoxylan (GX) extracted from aspen wood using subcritical water and alkaline conditions. Enzymatic digestions of GX with ß-xylanases from glycosyl hydrolase (GH) families GH10, GH11 and GH30 generated xylo-oligosaccharides with controlled structures amenable for mass spectrometric glycan sequencing. We identified the occurrence of intramolecular motifs in aspen GX with block repeats of even glucuronidation (every 2 xylose units) and consecutive glucuronidation, which are unique features for hardwood xylans. The acetylation pattern of aspen GX shows major domains with evenly-spaced decorations, together with minor stretches of highly acetylated domains. These heterogenous patterns of GX can be correlated with its extractability and with its potential interaction with lignin and cellulose. Our study provides new insights into the molecular structure of xylan in hardwood species, which has fundamental implications for overcoming lignocellulose recalcitrance during biochemical conversion.
Assuntos
Populus , Madeira , Xilanos , Xilanos/química , Xilanos/metabolismo , Madeira/química , Populus/química , Acetilação , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/metabolismo , Endo-1,4-beta-Xilanases/genética , Lignina/química , Celulose/química , Celulose/metabolismoRESUMO
Polysaccharide structural complexity not only influences cell wall strength and extensibility but also hinders pathogenic and biotechnological attempts to saccharify the wall. In certain species and tissues, glucuronic acid side groups on xylan exhibit arabinopyranose or galactose decorations whose genetic and evolutionary basis is completely unknown, impeding efforts to understand their function and engineer wall digestibility. Genetics and polysaccharide profiling were used to identify the responsible loci in Arabidopsis and Eucalyptus from proposed candidates, while phylogenies uncovered a shared evolutionary origin. GH30-family endo-glucuronoxylanase activities were analysed by electrophoresis, and their differing specificities were rationalised by phylogeny and structural analysis. The newly identified xylan arabinopyranosyltransferases comprise an overlooked subfamily in the GT47-A family of Golgi glycosyltransferases, previously assumed to comprise mainly xyloglucan galactosyltransferases, highlighting an unanticipated adaptation of both donor and acceptor specificities. Further neofunctionalisation has produced a Myrtaceae-specific xylan galactosyltransferase. Simultaneously, GH30 endo-glucuronoxylanases have convergently adapted to overcome these decorations, suggesting a role for these structures in defence. The differential expression of glucuronoxylan-modifying genes across Eucalyptus tissues, however, hints at further functions. Our results demonstrate the rapid adaptability of biosynthetic and degradative carbohydrate-active enzyme activities, providing insight into plant-pathogen interactions and facilitating plant cell wall biotechnological utilisation.
Assuntos
Arabidopsis , Parede Celular , Eucalyptus , Filogenia , Xilanos , Xilanos/metabolismo , Parede Celular/metabolismo , Arabidopsis/genética , Arabidopsis/enzimologia , Eucalyptus/genética , Eucalyptus/metabolismo , Hidrolases/metabolismo , Hidrolases/genética , Adaptação Fisiológica/genética , Glicosiltransferases/metabolismo , Glicosiltransferases/genética , Evolução MolecularRESUMO
A comparative analysis of chemical, functional, and digestive parameters was conducted on five new barley genotypes designed for food purposes, differing in starch type, ß-glucans, and arabinoxylan content. Both whole and pearled grain flours were examined. Amylose exhibited positive correlations with least gelation capacity (r = 0.60), gelation temperature (r = 0.90), and resistant starch (r = 0.80). Waxy varieties showed greater water-holding capacity, viscosity, and rapid digestibility compared to normal and high-amylose varieties. Pearling (10%) decreased arabinoxylans by 48% and proteins by 7%, while increasing ß-glucans by 8% and starch by 13%. Additionally, pearling improved viscosity and hydration parameters across varieties. This allowed normal and high-amylose genotypes to enhance their functional properties and nutritional value through increased ß-glucan and resistant starch content. This exploration advances the understanding of barley's functional attributes for food industry and underscores the potential of pearling to augment consumer nutritional value and health-promoting properties.