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1.
Food Chem ; 366: 130544, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34314932

RESUMO

The potential of travelling wave ion mobility spectroscopy in combination with collision induced dissociation tandem mass spectrometry (CID-TWIMS-MS/MS) to separate cereal-derived isomeric arabinoxylan-oligosaccharides (A)XOS was investigated. Three trisaccharide, four tetrasaccharide, and four pentasaccharide (A)XOS isomers were analyzed by positive and negative ionization TWIMS-MS and CID-TWIMS-MS/MS. The tri- and pentasaccharide isomers were distinguishable by the ATDs of the precursor ions. The CID-TWIMS-MS/MS could separate most of the isomeric fragment ions produced from tetra- and pentasaccharide (A)XOS. Finally, the base peak mobility spectrum is introduced as a practical tool for (A)XOS fingerprinting.


Assuntos
Grão Comestível , Espectrometria de Massas em Tandem , Espectrometria de Mobilidade Iônica , Oligossacarídeos , Xilanos
2.
Int J Mol Sci ; 22(17)2021 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-34502129

RESUMO

Size control is a fundamental question in biology, showing incremental complexity in plants, whose cells possess a rigid cell wall. The phytohormone auxin is a vital growth regulator with central importance for differential growth control. Our results indicate that auxin-reliant growth programs affect the molecular complexity of xyloglucans, the major type of cell wall hemicellulose in eudicots. Auxin-dependent induction and repression of growth coincide with reduced and enhanced molecular complexity of xyloglucans, respectively. In agreement with a proposed function in growth control, genetic interference with xyloglucan side decorations distinctly modulates auxin-dependent differential growth rates. Our work proposes that auxin-dependent growth programs have a spatially defined effect on xyloglucan's molecular structure, which in turn affects cell wall mechanics and specifies differential, gravitropic hypocotyl growth.


Assuntos
Glucanos/metabolismo , Ácidos Indolacéticos/metabolismo , Células Vegetais/metabolismo , Desenvolvimento Vegetal , Fenômenos Fisiológicos Vegetais , Xilanos/metabolismo , Arabidopsis/fisiologia , Parede Celular/metabolismo , Imunofluorescência , Regulação da Expressão Gênica de Plantas , Glucanos/química , Ervilhas/fisiologia , Transdução de Sinais , Xilanos/química
3.
Bioresour Technol ; 341: 125787, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34419877

RESUMO

Full utilization of lignocellulose is critical for its biorefinery development. In this study, a sustainable biorefinery process based upon poplar sawdust was established using sequential hydrothermal and deep eutectic solvent treatment (HP-DES). Results showed that single hydrothermal pretreatment (HP) could produce 53.2% xylo-oligosaccharides (XOS) (based on raw xylan), while the enzymatic digestibility was low. Conversely, single DES treatment achieved effective enzymatic digestibility but low XOS yields. As compared to HP, both DES treatment and HP-DES showed high selectivity for lignin removal and high glucose yield. Surprisingly, most of HP-DES residues had obviously lower enzymatic digestibilities than those of single DES residues. This was mainly explained by the differences of the surface lignin contents between DES and HP-DES residues. Moreover, nearly complete enzymatic hydrolysis of HP-DES residues was achieved with the addition of bovine serum albumin. This work demonstrated this HP-DES yielded XOS, fermentable sugar, and pure lignin with high processibility.


Assuntos
Lignina , Populus , Hidrólise , Oligossacarídeos , Solventes , Xilanos
4.
Bioresour Technol ; 341: 125817, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34454236

RESUMO

Lipids accumulated in the vegetative tissues of cellulosic feedstocks can be a potential raw material for biodiesel and bioethanol production. In this work, bagasse of genetically engineered sorghum was subjected to liquid hot-water pretreatment at 170, 180, and 190 °C for different reaction time. Under the optimal pretreatment condition (170 °C, 20 min), the residue was enriched in glucan (57.39 ± 2.63 % w/w) and xylan (13.38 ± 0.49 % w/w). The total lipid content of the pretreated residue was 6.81% w/w, similar to that observed in untreated bagasse (6.30% w/w). Pretreatment improved the enzymatic digestibility of bagasse, allowing a recovery of 79% w/w and 86% w/w of glucose and xylose, respectively. The pretreatment and enzymatic saccharification resulted in a 2-fold increase in total lipid in enzymatic residue compared to the original bagasse. Thus, pretreatment and enzymatic hydrolysis enabled high sugar recovery while concentrating triglycerides and free fatty acids in the residue.


Assuntos
Açúcares , Xilose , Hidrólise , Lipídeos , Xilanos
5.
J Agric Food Chem ; 69(31): 8838-8849, 2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34339210

RESUMO

Xyloglucans are the dominant hemicelluloses in the primary cell wall of dicotyledonous plants, fulfilling numerous functions. However, routine methods of cell wall analytical chemistry such as methylation analysis are time-consuming and often not adequate to capture the structural diversity of xyloglucans. Here, a xyloglucan profiling method based on the enzymatic release of xyloglucan oligosaccharides by a xyloglucan-specific endo-ß-(1→4)-glucanase and subsequent analysis of these oligosaccharides by high-performance anion-exchange chromatography (HPAEC) with parallel pulsed amperometric and mass spectrometric detection was developed. For this purpose, a set of 23 authentic xyloglucan oligosaccharides was generated, structurally characterized by mass spectrometry and NMR spectroscopy, and established as analytical standard compounds. Coupling of HPAEC with parallel electrochemical and MS detection was demonstrated to be an excellent tool to analyze xyloglucan-derived oligosaccharides. The applicability of the method was demonstrated by characterizing the xyloglucan architecture from a set of nine economically relevant food plants from the botanical orders Caryophyllales (rhubarb, buckwheat, amaranth, and quinoa), Cucurbitales (Hokkaido squash), Laurales (avocado), Myrtales (pomegranate), and Sapindales (mango and orange) for the first time. In future studies, this method can ideally be used to monitor structural alterations of xyloglucans as a result of genetic engineering, plant/tissue maturation, and processing of plant material.


Assuntos
Plantas Comestíveis , Xilanos , Ânions , Cromatografia , Glucanos , Espectrometria de Massas , Oligossacarídeos , Polissacarídeos
6.
Biomacromolecules ; 22(9): 3810-3818, 2021 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-34347473

RESUMO

Xylan-based films have great potential to replace petroleum-based polymers used for packaging and coatings due to their excellent biocompatibility, biodegradability, and good gas barrier properties. However, fabricating a xylan-based film with flexible, transparent, water-proof, and excellent mechanical properties is an enormous challenge. Herein, we manufactured a series of degradable films with adjustable properties via solution-casting using a water-soluble xylan derivative. This is the first report of a pure xylan-based film with high performance, requiring no additives. The tensile strength of the xylan-based film could be controlled by adjusting the aldehyde content, which varied from 105.0 to 132.6 MPa. The smallest initial water contact angle of the xylan-based films is 93.26°, indicating that these films are hydrophobic. This work shows a simple and viable route toward manufacturing xylan-based films with high tensile strength, flexibility, and transparency, which can be used for packaging materials and coatings.


Assuntos
Polímeros , Xilanos , Interações Hidrofóbicas e Hidrofílicas , Resistência à Tração , Água
7.
FEMS Microbiol Ecol ; 97(9)2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34415012

RESUMO

SAR92 is one of the few examples of a widely distributed, abundant oligotroph that can be cultivated to study pathways of carbon oxidation in ocean systems. Genomic evidence for SAR92 suggests that this gammaproteobacterium might be a primary consumer of polysaccharides in the epipelagic zone, its main habitat. Here, we investigated cell growth, polysaccharide utilization gene expression, and carbohydrate-active enzyme abundance of a culturable SAR92 strain, HTCC2207, grown with different polysaccharides. Xylan and laminarin, two polysaccharides mainly produced by phytoplankton, supported the growth of HTCC2207 better than other polysaccharides. HTCC2207 possessed polysaccharide utilization loci (PULs) consisting of TonB-dependent receptor (TBDR) and glycoside hydrolase (GH) family genes. GH genes such as GH17 and GH3 presented no substrate-specificity and were induced by different sugar substrates, while expressions of GH16, GH10 and GH30 were enhanced in the glucose-treatment but suppressed in the polysaccharide-treatment, indicating complex polysaccharide utilization by HTCC2207. Metabolic pathways for laminarin and xylan were re-constructed in HTCC2207 based on the PULs genes and other predicted carbohydrate-active enzymes. This study reveals features of the epipelagic niche of SAR92 and provide insight into the biogeochemical cycling of labile, high-molecular carbohydrate compounds in the surface ocean.


Assuntos
Gammaproteobacteria , Polissacarídeos , Fitoplâncton , Especificidade por Substrato , Xilanos
8.
Molecules ; 26(15)2021 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-34361682

RESUMO

Catalytic properties of GH30 xylanases belonging to subfamilies 7 and 8 were compared on glucuronoxylan, modified glucuronoxylans, arabinoxylan, rhodymenan, and xylotetraose. Most of the tested bacterial GH30-8 enzymes are specific glucuronoxylanases (EC 3.2.1.136) requiring for action the presence of free carboxyl group of MeGlcA side residues. These enzymes were not active on arabinoxylan, rhodymenan and xylotetraose, and conversion of MeGlcA to its methyl ester or its reduction to MeGlc led to a remarkable drop in their specific activity. However, some GH30-8 members are nonspecific xylanases effectively hydrolyzing all tested substrates. In terms of catalytic activities, the GH30-7 subfamily is much more diverse. In addition to specific glucuronoxylanases, the GH30-7 subfamily contains nonspecific endoxylanases and predominantly exo-acting enzymes. The activity of GH30-7 specific glucuronoxylanases also depend on the presence of the MeGlcA carboxyl, but not so strictly as in bacterial enzymes. The modification of the carboxyl group of glucuronoxylan had only weak effect on the action of predominantly exo-acting enzymes, as well as nonspecific xylanases. Rhodymenan and xylotetraose were the best substrates for exo-acting enzymes, while arabinoxylan represented hardly degradable substrate for almost all tested GH30-7 enzymes. The results expand current knowledge on the catalytic properties of this relatively novel group of xylanases.


Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Proteínas Fúngicas/metabolismo , Fungos/enzimologia , Xilosidases/metabolismo , Catálise , Hidrólise , Especificidade por Substrato , Xilanos/metabolismo
9.
Molecules ; 26(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34361767

RESUMO

This study describes the catalytic properties of a GH30_7 xylanase produced by the fungus Talaromyces leycettanus. The enzyme is an ando-ß-1,4-xylanase, showing similar specific activity towards glucuronoxylan, arabinoxylan, and rhodymenan (linear ß-1,3-ß-1,4-xylan). The heteroxylans are hydrolyzed to a mixture of linear as well as branched ß-1,4-xylooligosaccharides that are shorter than the products generated by GH10 and GH11 xylanases. In the rhodymenan hydrolyzate, the linear ß-1,4-xylooligosaccharides are accompanied with a series of mixed linkage homologues. Initial hydrolysis of glucuronoxylan resembles the action of other GH30_7 and GH30_8 glucuronoxylanases, resulting in a series of aldouronic acids of a general formula MeGlcA2Xyln. Due to the significant non-specific endoxylanase activity of the enzyme, these acidic products are further attacked in the unbranched regions, finally yielding MeGlcA2Xyl2-3. The accommodation of a substituted xylosyl residue in the -2 subsite also applies in arabinoxylan depolymerization. Moreover, the xylose residue may be arabinosylated at both positions 2 and 3, without negatively affecting the main chain cleavage. The catalytic properties of the enzyme, particularly the great tolerance of the side-chain substituents, make the enzyme attractive for biotechnological applications. The enzyme is also another example of extraordinarily great catalytic diversity among eukaryotic GH30_7 xylanases.


Assuntos
Endo-1,4-beta-Xilanases/metabolismo , Proteínas Fúngicas/metabolismo , Talaromyces/enzimologia , Xilanos/metabolismo , Sequência de Aminoácidos , Arabinose/química , Arabinose/metabolismo , Sequência de Carboidratos , Endo-1,4-beta-Xilanases/genética , Proteínas Fúngicas/genética , Expressão Gênica , Glucuronatos/química , Glucuronatos/metabolismo , Hidrólise , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Talaromyces/química , Talaromyces/genética , Xilanos/química
10.
Bioresour Technol ; 340: 125695, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34364087

RESUMO

Hydrogen bond donor (HBD) in ChCl-based deep eutectic solvent (DESs) had significant influence on the Sorghum straw (SS) pretreatment. Lactic acid (LAC) was chosen as the appropriate HBD for preparing ChCl-based DES to pretreat Sorghum straw (SS). Furthermore, sequential pretreatment with dilute sodium hydroxide (0.75 wt%) for 1 h at 121 °C and ChCl:LAC soaking at 140 °C for 40 min was applied to pretreat SS for removing lignin (78.4%) and xylan (67.6%). Hydrolysis for 72 h, the reducing sugar yield reached 94.9%. Moreover, relationships of delignification and xylan removal with saccharification were explored after pretreatment. Finally, the fermentability of SS-hydrolysates was verified by bioethanol fermentation by S. cerevissiae with the yield of 0.45 g ethanol/g glucose. No significant inhibition was observed on ethanol fermentation. Obviously, establishment of high-efficient combination pretreatment with alkali extraction and ChCl:LAC soaking was successfully demonstrated for enhancing enzymatic saccharification of SS.


Assuntos
Sorghum , Álcalis , Lignina , Solventes , Xilanos
11.
Bioresour Technol ; 340: 125732, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34426240

RESUMO

The ancestor of ß-1,3-xylanases (AncXyl09) were reconstructed by the optimized ancestral sequences reconstruction strategy to solve the poor catalytic performances of existing ß-1,3-xylanases. The results showed that the half-life at 50 °C was 65.08 h, indicating good thermostability. The large number of hydrogen bonds and the disulfide bonds were the major attributes related with the thermal stability of Anxyl09. Interestingly, AncXyl09 could hydrolyze lichen besides the original substrate of ß-1, 3-xylan, which is the first reported ß-1,3-xylanase with substrate promiscuity. Moreover, the hydrolytic products are mainly disaccharides, the content of ß-1,3-xylobiose and lichoridiose more than 70% as determined by high performance liquid chromatography (HPLC), which could significantly facilitate the separation and purification of oligosaccharides. The successful design of AncXyl09 was the representative of the semi-rationally engineered ß-1, 3-xylanase, which will shield a new light on the ß-1,3-xylanase engineering, active oligosaccharide preparation and marine algae resource utilization.


Assuntos
Endo-1,4-beta-Xilanases , Xilanos , Endo-1,4-beta-Xilanases/metabolismo , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Especificidade por Substrato , Temperatura
12.
Food Res Int ; 147: 110466, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34399464

RESUMO

Dietary fiber plays an important role in the prevention of colorectal cancer, and arabinoxylans are an important source of this in grains, with some studies reporting the inhibition of cancer cell growth. However, very few studies have been conducted on this, and most previous studies have used oligosaccharides derived from arabinoxylans of specific molecular weight. The aim of this work is to extract, isolate, and analyze arabinoxylans from two different Argentinian genotypes of wheat (hard and soft) and study if they have the capacity to decrease the cellular viability of a colon cancer line (HCT-116). To determine whether the molecular size influences the inhibition of HCT-116 cell viability, specific hydrolysis was performed with endoxylanase, and the cells were exposed to the hydrolyzed arabinoxylans. The arabinoxylans treatment resulted in HCT-116 cell viability of 74% for the soft genotype and 64% for the hard genotype in comparison to nontreated cells. Hydrolyzed-arabinoxylans result in HCT-116 cell viability of 68% for soft and 36% for hard genotypes (the lowest IC50 values) compared to nontreated cells. More importantly, no decrease after the arabinoxylans treatment was observed in the viability of murine noncancer cells known to rapidly respond to polysaccharide presence. The arabinoxylans from hard wheat showed more disubstituted xylose and α-1,2/α-1,3 linkages than the arabinoxylans from soft wheat, the possible cause for showing the best in vitro biological effect. The results showed other beneficial effects than the prebiotic ones and support the use of enzymatic treatment to increase the biological impacts of arabinoxylans.


Assuntos
Triticum , Xilanos , Animais , Fibras na Dieta , Células HCT116 , Humanos , Camundongos
13.
Appl Microbiol Biotechnol ; 105(18): 6759-6778, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34458936

RESUMO

The genus Cohnella belongs to a group of Gram-positive endospore-forming bacteria within the Paenibacillaceae family. Although most species were described as xylanolytic bacteria, the literature still lacks some key information regarding their repertoire of xylan-degrading enzymes. The whole genome sequence of an isolated xylan-degrading bacterium Cohnella sp. strain AR92 was found to contain five genes encoding putative endo-1,4-ß-xylanases, of which four were cloned, expressed, and characterized to better understand the contribution of the individual endo-xylanases to the overall xylanolytic properties of strain AR92. Three of the enzymes, CoXyn10A, CoXyn10C, and CoXyn11A, were shown to be effective at hydrolyzing xylans-derived from agro-industrial, producing oligosaccharides with substrate conversion values of 32.5%, 24.7%, and 10.6%, respectively, using sugarcane bagasse glucuronoarabinoxylan and of 29.9%, 19.1%, and 8.0%, respectively, using wheat bran-derived arabinoxylan. The main reaction products from GH10 enzymes were xylobiose and xylotriose, whereas CoXyn11A produced mostly xylooligosaccharides (XOS) with 2 to 5 units of xylose, often substituted, resulting in potentially prebiotic arabinoxylooligosaccharides (AXOS). The endo-xylanases assay displayed operational features (temperature optima from 49.9 to 50.4 °C and pH optima from 6.01 to 6.31) fitting simultaneous xylan utilization. Homology modeling confirmed the typical folds of the GH10 and GH11 enzymes, substrate docking studies allowed the prediction of subsites (- 2 to + 1 in GH10 and - 3 to + 1 in GH11) and identification of residues involved in ligand interactions, supporting the experimental data. Overall, the Cohnella sp. AR92 endo-xylanases presented significant potential for enzymatic conversion of agro-industrial by-products into high-value products.Key points• Cohnella sp. AR92 genome encoded five potential endo-xylanases.• Cohnella sp. AR92 enzymes produced xylooligosaccharides from xylan, with high yields.• GH10 enzymes from Cohnella sp. AR92 are responsible for the production of X2 and X3 oligosaccharides.• GH11 from Cohnella sp. AR92 contributes to the overall xylan degradation by producing substituted oligosaccharides.


Assuntos
Bacillales , Saccharum , Endo-1,4-beta-Xilanases/genética , Hidrólise , Oligossacarídeos , Xilanos
14.
J Agric Food Chem ; 69(31): 8610-8624, 2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34324332

RESUMO

ß-1,4-Xylan is the main component of hemicelluloses in land plant cell walls, whereas ß-1,3-xylan is widely found in seaweed cell walls. Complete hydrolysis of xylan requires a series of synergistically acting xylanases. High-saline environments, such as saline-alkali lands and oceans, frequently occur in nature and are also involved in a broad range of various industrial processes. Thus, salt-tolerant xylanases may contribute to high-salt and marine food processing, aquatic feed production, industrial wastewater treatment, saline-alkali soil improvement, and global carbon cycle, with great commercial and environmental benefits. This review mainly introduces the definition, sources, classification, biochemical and molecular characteristics, adaptation mechanisms, and biotechnological applications of salt-tolerant xylanases. The scope of development for salt-tolerant xylanases is also discussed. It is anticipated that this review would serve as a reference for further development and utilization of salt-tolerant xylanases and other salt-tolerant enzymes.


Assuntos
Endo-1,4-beta-Xilanases , Alga Marinha , Biotecnologia , Hidrólise , Xilanos
15.
Nutrients ; 13(6)2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-34203983

RESUMO

Arabinoxylan (AX) is a structural polysaccharide found in wheat, rice and other cereal grains. Diets high in AX-containing fiber may promote gut health in obesity through prebiotic function. Thus, the impact of soluble AX isolated from rice bran fiber on human gut microbiota phylogenetic composition and short-chain fatty acid (SCFA) production patterns from normal-weight and overweight/obese subjects was investigated through in vitro fecal fermentation. Results showed that rice bran arabinoxylan modified the microbiota in fecal samples from both weight classes compared to control, significantly increasing Collinsella, Blautia and Bifidobacterium, and decreasing Sutterella, Bilophila and Parabacteroides. Rice bran AX also significantly increased total and individual SCFA contents (p < 0.05). This study suggests that rice bran AX may beneficially impact gut health in obesity through prebiotic activities.


Assuntos
Fezes/microbiologia , Fermentação , Obesidade/microbiologia , Oryza/química , Xilanos/metabolismo , Adulto , Bactérias/classificação , Dieta , Carboidratos da Dieta , Fibras na Dieta , Grão Comestível , Ácidos Graxos Voláteis , Feminino , Microbioma Gastrointestinal , Humanos , Masculino , Sobrepeso , Filogenia , Prebióticos , Triticum , Xilanos/isolamento & purificação
16.
Enzyme Microb Technol ; 149: 109834, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34311879

RESUMO

The goal of this work was the autodisplay of the endo ß-1,4-xylanase (XynA) from Clostridium cellulovorans in Escherichia coli using the AIDA system to carry out whole-cell biocatalysis and hydrolysate xylans. For this, pAIDA-xynA vector containing a synthetic xynA gene was fused to the signal peptide of the toxin subunit B Vibro cholere (ctxB) and the auto-transporter of the synthetic aida gene, which encodes for the connector peptide and ß-barrel of the auto-transporter (AT-AIDA). E. coli TOP10 cells were transformed and the biocatalyst was characterized using beechwood xylans as substrate. Optimal operational conditions were temperature of 55 °C and pH 6.5, and the Michaelis-Menten catalytic constants Vmax and Km were 149 U/gDCW and 6.01 mg/mL, respectively. Xylanase activity was inhibited by Cu2+, Zn2+ and Hg2+ as well as EDTA, detergents, and organic acids, and improved by Ca2+, Co2+ and Mn2+ ions. Ca2+ ion strongly enhanced the xylanolytic activity up to 2.4-fold when 5 mM CaCl2 were added. Also, Ca2+ improved enzyme stability at 60 and 70 °C. Results suggest that pAIDA-xynA vector has the ability to express functional xylanase to perform whole-cell biocatalysis in order to hydrolysate xylans from hemicellulose feedstock.


Assuntos
Clostridium cellulovorans , Xilanos , Clostridium cellulovorans/metabolismo , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Temperatura
17.
Enzyme Microb Technol ; 149: 109854, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34311891

RESUMO

Xylanases are categorized into different family groups, two of which are glycoside hydrolases 10 (GH10) and 11 (GH11) families. These well-characterized xylanases demonstrate different modes of action in hydrolysis of xylans. Imitating certain types of microorganisms to produce bifunctional enzymes such as engineered xylanases has gained considerable attention among researchers. In this study, a recombinant chimeric enzyme (X11-10) was designed by fusing two thermostable xylanases through a peptide linker. The recombinant parental enzymes, xylanase 10 from fungus Bispora sp. MEY-1 (X10) and xylanase 11 from bacterium Thermobacillus xylanilyticus (X11), and their chimera were successfully expressed in Pichia pastoris (P. pastoris), purified, and characterized. Being active over a wide pH range, X11-10 chimera showed higher thermal stability, possessed a lower Km, and a higher catalytic efficiency (kcat/Km) in comparison to the parental enzymes. Also, molecular dynamics simulation (MDS) of X11-10 revealed that its active site residues were free to interact with substrate. This novel chimeric xylanase may have potential applications in different industrial processes since it can substitute two separate enzymes and therefore minimize the production costs.


Assuntos
Ascomicetos , Xilanos , Ascomicetos/metabolismo , Bacillales , Quimera/metabolismo , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Estabilidade Enzimática , Glicosídeo Hidrolases , Proteínas Recombinantes de Fusão/genética , Saccharomycetales , Especificidade por Substrato
18.
Nat Commun ; 12(1): 4049, 2021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-34193873

RESUMO

Xyloglucans are highly substituted and recalcitrant polysaccharides found in the primary cell walls of vascular plants, acting as a barrier against pathogens. Here, we reveal that the diverse and economically relevant Xanthomonas bacteria are endowed with a xyloglucan depolymerization machinery that is linked to pathogenesis. Using the citrus canker pathogen as a model organism, we show that this system encompasses distinctive glycoside hydrolases, a modular xyloglucan acetylesterase and specific membrane transporters, demonstrating that plant-associated bacteria employ distinct molecular strategies from commensal gut bacteria to cope with xyloglucans. Notably, the sugars released by this system elicit the expression of several key virulence factors, including the type III secretion system, a membrane-embedded apparatus to deliver effector proteins into the host cells. Together, these findings shed light on the molecular mechanisms underpinning the intricate enzymatic machinery of Xanthomonas to depolymerize xyloglucans and uncover a role for this system in signaling pathways driving pathogenesis.


Assuntos
Parede Celular/metabolismo , Citrus/microbiologia , Glucanos/metabolismo , Glicosídeo Hidrolases/metabolismo , Fatores de Virulência/genética , Xanthomonas/metabolismo , Xilanos/metabolismo , Proteínas de Bactérias/metabolismo , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Ativação Transcricional , Sistemas de Secreção Tipo III/metabolismo , Fatores de Virulência/metabolismo , Xanthomonas/genética , Xanthomonas/patogenicidade
19.
New Phytol ; 231(5): 1720-1733, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34086997

RESUMO

Wood of coniferous trees (softwood), is a globally significant carbon sink and an important source of biomass. Despite that, little is known about the genetic basis of softwood cell wall biosynthesis. Branching of xylan, one of the main hemicelluloses in softwood secondary cell walls, with glucuronic acid (GlcA) is critical for biomass recalcitrance. Here, we investigate the decoration patterns of xylan by conifer GlucUronic acid substitution of Xylan (GUX) enzymes. Through molecular phylogenetics we identify two distinct conifer GUX clades. Using transcriptional profiling we show that the genes are preferentially expressed in secondary cell wall forming tissues. With in vitro and in planta assays we demonstrate that conifer GUX enzymes from both clades are active glucuronyltransferases. Conifer GUX enzymes from each clade have different specific activities. While members of clade one add evenly spaced GlcA branches, the members of clade two are also capable of glucuronidating two consecutive xyloses. Importantly, these types of xylan patterning are present in softwood. As xylan patterning might modulate xylan-cellulose and xylan-lignin interactions, our results further the understanding of softwood cell wall biosynthesis and provide breeding or genetic engineering targets that can be used to modify softwood properties.


Assuntos
Arabidopsis , Traqueófitas , Parede Celular , Ácido Glucurônico , Melhoramento Vegetal , Traqueófitas/genética , Xilanos
20.
Int J Biol Macromol ; 185: 40-48, 2021 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-34144065

RESUMO

It is well known that the chemical structure of polysaccharides is important to their final biological effect. In this study we investigated the cytotoxic effect of xyloglucan from Copaifera langsdorffii seeds (XGC) and its complex with oxovanadium (XGC:VO) on hepatocellular carcinoma cells (HepG2). After 72 h of incubation, XGC and XGC:VO (200 µg/mL) reduced cell viability in ~20% and ~40%, respectively. At same conditions, only XGC:VO increased in ~20% the LDH enzyme release. In permeabilized cells, incubated with XGC and XGC:VO (200 µg/mL) for 72 h, NADH oxidase activity was reduced by ~45% with XGC and XGC:VO. The succinate oxidase activity was reduced by ~35% with XGC and ~65% with XGC:VO, evidencing that polysaccharide complexation with vanadium could intensify its effects on the respiratory chain. According to this result, the mitochondrial membrane potential was also reduced by ~9% for XGC and ~30% for XGC:VO, when compared to the control group. Interestingly, ATP levels were more elevated for XGC:VO in respect to XGC, probably due the enhance in glycolytic flux evidenced by increased levels of lactate. These results show that the xyloglucan complexation with oxovanadium (IV/V) potentiates the cytotoxic effect of the native polysaccharide, possibly by impairment of oxidative phosphorylation.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Hepatocelular/metabolismo , Fabaceae/química , Glucanos/farmacologia , Neoplasias Hepáticas/metabolismo , Vanadatos/química , Xilanos/farmacologia , Antineoplásicos/química , Carcinoma Hepatocelular/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glucanos/química , Células Hep G2 , Humanos , L-Lactato Desidrogenase/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Oxirredutases/metabolismo , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Xilanos/química
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