RESUMO
The marine environment, contains plentiful renewable resources, e.g. macroalgae with unique polysaccharides, motivating search for enzymes from marine microorganisms to explore conversion possibilities of the polysaccharides. In this study, the first GH17 glucanosyltransglycosylase, MlGH17B, from a marine bacterium (Muricauda lutaonensis), was characterized. The enzyme was moderately thermostable with Tm at 64.4 °C and 73.2 °C, but an activity optimum at 20 °C, indicating temperature sensitive active site interactions. MlGH17B uses ß-1,3 laminari-oligosaccharides with a degree of polymerization (DP) of 4 or higher as donors. Two glucose moieties (bound in the aglycone +1 and +2 subsites) are cleaved off from the reducing end of the donor while the remaining part (bound in the glycone subsites) is transferred to an incoming ß-1,3 glucan acceptor, making a ß-1,6-linkage, thereby synthesizing branched or kinked oligosaccharides. Synthesized oligosaccharides up to DP26 were detected by mass spectrometry analysis, showing that repeated transfer reactions occurred, resulting in several ß-1,6-linked branches. The modeled structure revealed an active site comprising five subsites: three glycone (-3, -2 and -1) and two aglycone (+1 and +2) subsites, with significant conservation of substrate interactions compared to the only crystallized 1,3-ß-glucanosyltransferase from GH17 (RmBgt17A from the compost thriving fungus Rhizomucor miehei), suggesting a common catalytic mechanism, despite different phylogenetic origin, growth environment, and natural substrate. Both enzymes lacked the subdomain extending the aglycone subsites, found in GH17 endo-ß-glucanases from plants, but this extension was also missing in bacterial endoglucanases (modeled here), showing that this feature does not distinguish transglycosylation from hydrolysis, but may rather relate to phylogeny.
Assuntos
Flavobacteriaceae , Oligossacarídeos , Filogenia , Oligossacarídeos/química , Polissacarídeos , Especificidade por SubstratoRESUMO
An uncharacterized gene encoding a glycoside hydrolase family 43-like enzyme from Clostridium boliviensis strain E-1 was identified from genomic sequence data, and the encoded enzyme, CbE1Xyn43-l, was produced in Escherichia coli. CbE1Xyn43-l (52.9 kDa) is a two-domain endo-ß-xylanase consisting of a C-terminal CBM6 and a GH43-like catalytic domain. The positions of the catalytic dyad conserved in GH43, the catalytic base (Asp74), and proton donor (Glu240) were identified in alignments including GH43-enzymes of known 3D-structure from different subfamilies. CbE1Xyn43-l is active at pH 7.0-9.0, with optimum temperature at 65°C, and a more than 7 days' half-life in irreversible deactivation studies at this temperature. The enzyme hydrolyzed birchwood xylan, quinoa stalks glucuronoarabinoxylan, and wheat arabinoxylan with xylotriose and xylotetraose as major hydrolysis products. CbE1Xyn43-l also released xylobiose from pNPX2 with low turnover (kcat of 0.044 s-1) but was inactive on pNPX, showing that a degree of polymerization of three (DP3) was the smallest hydrolyzable substrate. Divalent ions affected the specific activity on xylan substrates, which dependent on the ion could be increased or decreased. In conclusion, CbE1Xyn43-l from C. boliviensis strain E-1 is the first characterized member of a large group of homologous hypothetical proteins annotated as GH43-like and is a thermostable endo-xylanase, producing xylooligosaccharides of high DP (xylotriose and xylotetraose) producer. IMPORTANCE: The genome of Clostridium boliviensis strain E-1 encodes a number of hypothetical enzymes, annotated as glycoside hydrolase-like but not classified in the Carbohydrate Active Enzyme Database (CAZy). A novel thermostable GH43-like enzyme is here characterized as an endo-ß-xylanase of interest in the production of prebiotic xylooligosaccharides (XOs) from different xylan sources. CbE1Xyn43-l is a two-domain enzyme composed of a catalytic GH43-l domain and a CBM6 domain, producing xylotriose as main XO product. The enzyme has homologs in many related Clostridium strains which may indicate a similar function and be a previously unknown type of endo-xylanase in this evolutionary lineage of microorganisms.
Assuntos
Glucuronatos , Glicosídeo Hidrolases , Oligossacarídeos , Xilanos , Xilanos/metabolismo , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Especificidade por Substrato , Clostridium/genética , Clostridium/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Hidrólise , Estabilidade Enzimática , Concentração de Íons de HidrogênioRESUMO
Sulfation is gaining increased interest due to the role of sulfate in the bioactivity of many polysaccharides of marine origin. Hence, sulfatases, enzymes that control the degree of sulfation, are being more extensively researched. In this work, a novel sulfatase (SulA1) encoded by the gene sulA1 was characterized. The sulA1-gene is located upstream of a chondroitin lyase encoding gene in the genome of the marine Arthrobacter strain (MAT3885). The sulfatase was produced in Escherichia coli. Based on the primary sequence, the enzyme is classified under sulfatase family 1 and the two catalytic residues typical of the sulfatase 1 family-Cys57 (post-translationally modified to formyl glycine for function) and His190-were conserved. The enzyme showed increased activity, but not improved stability, in the presence of Ca2+, and conserved residues for Ca2+ binding were identified (Asp17, Asp18, Asp277, and Asn278) in a structural model of the enzyme. The temperature and pH activity profiles (screened using p-nitrocatechol sulfate) were narrow, with an activity optimum at 40-50 °C and a pH optimum at pH 5.5. The Tm was significantly higher (67 °C) than the activity optimum. Desulfation activity was not detected on polymeric substrates, but was found on GalNAc4S, which is a sulfated monomer in the repeated disaccharide unit (GlcA-GalNAc4S) of, e.g., chondroitin sulfate A. The position of the sulA1 gene upstream of a chondroitin lyase gene and combined with the activity on GalNAc4S suggests that there is an involvement of the enzyme in the chondroitin-degrading cascade reaction, which specifically removes sulfate from monomeric GalNAc4S from chondroitin sulfate degradation products.
Assuntos
Arthrobacter , Sulfatos , Acetilgalactosamina , Sulfatases , Escherichia coli , Galactosamina , Condroitina Liases , Clonagem MolecularRESUMO
Enzymes' uncharacterised side activities can have significant effects on reaction products and yields. Hence, their identification and characterisation are crucial for the development of successful reaction systems. Here, we report the presence of feruloyl esterase activity in CtXyn5A from Acetivibrio thermocellus, besides its well-known arabinoxylanase activity, for the first time. Activity analysis of enzyme variants mutated in the catalytic nucleophile, Glu279, confirmed removal of all activity for E279A and E279L, and increased esterase activity while removing xylanase activity for E279S, thus allowing the proposal that both reaction types are catalysed in the same active site in two subsequential steps. The ferulic acid substituent is cleaved off first, followed by hydrolysis of the xylan backbone. The esterase activity on complex carbohydrates was found to be higher than that of a designated ferulic acid esterase (E-FAERU). Therefore, we conclude that the enzyme exhibits a dual function rather than an esterase side activity.
Assuntos
Hidrolases de Éster Carboxílico , Xilanos , Domínio Catalítico , Hidrolases de Éster Carboxílico/química , Especificidade por SubstratoRESUMO
Halophilic archaea are polyextremophiles with the ability to withstand fluctuations in salinity, high levels of ultraviolet radiation, and oxidative stress, allowing them to survive in a wide range of environments and making them an excellent model for astrobiological research. Natrinema altunense 4.1R is a halophilic archaeon isolated from the endorheic saline lake systems, Sebkhas, located in arid and semi-arid regions of Tunisia. It is an ecosystem characterized by periodic flooding from subsurface groundwater and fluctuating salinities. Here, we assess the physiological responses and genomic characterization of N. altunense 4.1R to UV-C radiation, as well as osmotic and oxidative stresses. Results showed that the 4.1R strain is able to survive up to 36% of salinity, up to 180 J/m2 to UV-C radiation, and at 50 mM of H2O2, a resistance profile similar to Halobacterium salinarum, a strain often used as UV-C resistant model. In order to understand the genetic determinants of N. altunense 4.1R survival strategy, we sequenced and analyzed its genome. Results showed multiple gene copies of osmotic stress, oxidative stress, and DNA repair response mechanisms supporting its survivability at extreme salinities and radiations. Indeed, the 3D molecular structures of seven proteins related to responses to UV-C radiation (excinucleases UvrA, UvrB, and UvrC, and photolyase), saline stress (trehalose-6-phosphate synthase OtsA and trehalose-phosphatase OtsB), and oxidative stress (superoxide dismutase SOD) were constructed by homology modeling. This study extends the abiotic stress range for the species N. altunense and adds to the repertoire of UV and oxidative stress resistance genes generally known from haloarchaeon.
Assuntos
Halobacteriaceae , Raios Ultravioleta , Ecossistema , Peróxido de Hidrogênio , Halobacteriaceae/genética , Estresse Oxidativo , GenômicaRESUMO
One of the indispensable applications of lipases in modification of oils and fats is the possibility to tailor the fatty acid content of triacylglycerols (TAGs), to meet specific requirements from various applications in food, nutrition, and cosmetic industries. Oleic acid (C18:1) and stearic acid (C18:0) are two common long fatty acids in the side chain of triglycerides in plant fats and oils that have similar chemical composition and structures, except for an unsaturated bond between C9 and C10 in oleic acid. Two lipases from Rhizomucor miehei (RML) and Rhizopus oryzae (ROL), show activity in reactions involving oleate and stearate, and share high sequence and structural identity. In this research, the preference for one of these two similar fatty acid side chains was investigated for the two lipases and was related to the respective enzyme structure. From transesterification reactions with 1:1 (molar ratio) mixed ethyl stearate (ES) and ethyl oleate (EO), both RML and ROL showed a higher activity towards EO than ES, but RML showed around 10% higher preference for ES compared with ROL. In silico results showed that stearate has a less stable interaction with the substrate binding crevice in both RML and ROL and higher tendency to freely move out of the substrate binding region, compared with oleate whose structure is more rigid due to the existence of the double bond. However, Trp88 from RML which is an Ala at the identical position in ROL shows a significant stabilization effect in the substrate interaction in RML, especially with stearate as a ligand.
Assuntos
Proteínas Fúngicas , Lipase , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Lipase/química , Lipase/genética , Simulação de Acoplamento Molecular , Ácidos Oleicos , Rhizomucor/enzimologia , Rhizopus oryzae/enzimologia , Análise de Sequência de Proteína , Estearatos , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Alkyl glycoside surfactants with elongated carbohydrate chains are useful in different applications due to their improved biocompatibility. Cyclodextrin glucanotransferases can catalyze the elongation process through the coupling reaction. However, due to the presence of a hydrophobic tail, the interaction between an alkyl glycoside acceptor and the active site residues is weaker than the interaction with maltooligosaccharides at the corresponding site. Here we report the mutations of F197, G263 and E266 near the acceptor subsites in the CGTase CspCGT13 from Carboxydocella sp. The results showed that substitutions of both F197 and G263 were important for the binding of acceptor substrate dodecyl maltoside during coupling reaction. The double mutant F197Y/G263A showed enhanced coupling activity and displayed a 2-fold increase of the primary coupling product using γ-cyclodextrin as donor when compared to wildtype CspCGT13. Disproportionation activity was also reduced, which was also the case for another double mutant (F197Y/E266A) that however not showed the corresponding increase in coupling. A triple mutant F197Y/G263A/E266A maintained the increase in primary coupling product (1.8-fold increase) using dodecyl maltoside as acceptor, but disproportionation was approximately at the same level as in the double mutants. In addition, hydrolysis of starch was slightly increased by the F197Y and G263A substitutions, indicating that interactions at both positions influenced the selectivity between glycosyl and alkyl moieties.
Assuntos
Glucosiltransferases/metabolismo , Glicosídeos/biossíntese , Engenharia de Proteínas , Bactérias Anaeróbias/enzimologia , Biologia Computacional , Glucosiltransferases/genética , Glicosídeos/química , Glicosídeos/genética , Modelos Moleculares , MutaçãoRESUMO
Prevotella copri is a bacterium that can be found in the human gastrointestinal tract (GIT). The role of P. copri in the GIT is unclear, and elevated numbers of the microbe have been reported both in dietary fiber-induced improvement in glucose metabolism but also in conjunction with certain inflammatory conditions. These findings raised our interest in investigating the possibility of P. copri to grow on xylan, and identify the enzyme systems playing a role in digestion of xylan-based dietary fibers. Two xylan degrading polysaccharide utilizing loci (PUL10 and 15) were found in the genome, with three and eight glycoside hydrolase (GH) -encoding genes, respectively. Three of them were successfully produced in Escherichia coli: One extracellular enzyme from GH43 (subfamily 12, in PUL10, 60 kDa) and two enzymes from PUL15, one extracellular GH10 (41 kDa), and one intracellular GH43 (subfamily 137 kDa). Based on our results, we propose that in PUL15, GH10 (1) is an extracellular endo-1,4-ß-xylanase, that hydrolazes mainly glucuronosylated xylan polymers to xylooligosaccharides (XOS); while, GH43_1 in the same PUL, is an intracellular ß-xylosidase, catalyzing complete hydrolysis of the XOS to xylose. In PUL10, the characterized GH43_12 is an arabinofuranosidase, with a role in degradation of arabinoxylan, catalyzing removal of arabinose-residues on xylan.
Assuntos
Glicosídeo Hidrolases/metabolismo , Polissacarídeos/metabolismo , Prevotella/química , Xilanos/metabolismo , Glicosídeo Hidrolases/química , Cinética , Modelos Moleculares , Polissacarídeos/química , Prevotella/metabolismo , Xilanos/químicaRESUMO
Modification of alkyl glycosides, to alter their properties and widen the scope of potential applications, is of considerable interest. Here, we report the synthesis of new anionic alkyl glycosides with long carbohydrate chains, using two different approaches: laccase/2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) oxidation of a long-carbohydrate-chain alkyl glycoside and cyclodextrin glucanotransferase (CGTase)-catalyzed elongation of anionic alkyl glycosides. The laccase/TEMPO oxidation of dodecyl ß- d-maltooctaoside proceeded efficiently with the formation of aldehyde and acid products. However, depolymerization occurred to a large extent, limiting the product yield and purity. On the other hand, CGTase-catalyzed coupling/disproportionation reactions with α-cyclodextrin and dodecyl ß- d-maltoside diuronic acid (DDM-2COOH) or octyl ß- d-glucuronic acid (OG-COOH) as substrates gave high conversions, especially when the CGTase Toruzyme was used. It was found that pH had a strong influence on both the enzyme activity and the acceptor specificity. With non-ionic substrates (dodecyl ß- d-maltoside and octyl ß- d-glucoside), Toruzyme exhibited high catalytic activity at pH 5-6, but for the acidic substrates (DDM-2COOH and OG-COOH) the activity was highest at pH 4. This is most likely due to the enzyme favoring the protonated forms of DDM-2COOH and OG-COOH, which exist at lower pH (pKa about 3).
Assuntos
Proteínas de Bactérias/química , Glucosiltransferases/química , Glicosídeos , Lacase/química , Paenibacillus/enzimologia , Thermoanaerobacter/enzimologia , Catálise , Glicosídeos/síntese química , Glicosídeos/química , OxirreduçãoRESUMO
Most macromolecular antimicrobials are ionic and thus lack miscibility/compatibility with nonionic substrate materials. In this context, nonionic hyperbranched polyesters (HBPs) with indole or isatin functionality were rationally designed, synthesized, and characterized. Antimicrobial disk diffusion assay indicated that these HBPs showed significant antibacterial activity against 8 human pathogenic bacteria compared to small molecules with indole or isatin groups. According to DSC measurements, up to 20% indole-based HBP is miscible with biodegradable polyesters (polyhydroxybutyrate or polycaprolactone), which can be attributed to the favorable hydrogen bonding between the N-H moiety of indole and the CâO of polyesters. HBPs with isatin or methylindole were completely immiscible with the same matrices. None of the HBPs leaked out from plastic matrix after being immersed in water for 5 days. The incorporation of indole into HBPs as well as small molecules facilitated their enzymatic degradation with PETase from Ideonella sakaiensis, while isatin had a complex impact. Molecular docking simulations of monomeric molecules with PETase revealed different orientations of the molecules at the active site due to the presence of indole or isatin groups, which could be related to the observed different enzymatic degradation behavior. Finally, biocompatibility analysis with a mammalian cell line showed the negligible cytotoxic effect of the fabricated HBPs.
Assuntos
Isatina , Animais , Antibacterianos , Burkholderiales , Humanos , Indóis , Isatina/farmacologia , Simulação de Acoplamento Molecular , Poliésteres , PolímerosRESUMO
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 , XilanosRESUMO
We report the facile synthesis of a series of indole-based hydroxyl-carboxylate (AB-type) monomers by a one-step procedure. These monomers were successfully polymerized by melt polycondensation to yield AB-type polyesters with a varied number of flexible methylene units in the backbones. These indole-based AB-polyesters showed decent thermal stability according to the TGA results (onset thermal degradation temperature of >330 °C), and their glass transition temperatures are dependent on the length of the methylene bridge (Tg ≈ 62-102 °C) according to the DSC results. Furthermore, DSC and WAXD measurements revealed that these polymers did not crystallize from melt, but the ones with flexible structures could crystallize from solution. Molecular docking simulations showed favorable interactions between indole-based polyesters and polyethylene terephthalate hydrolase (PETase) from Ideonella sakaiensis. This was corroborated by the experimental results, which indicated that the PETase enzyme has degrading activity on the indole-based AB polyesters except for the one with the highest degree of crystallinity.
Assuntos
Burkholderiales , Poliésteres , Indóis , Simulação de Acoplamento Molecular , PolietilenotereftalatosRESUMO
Hydrolysis of arabinoxylan (AX) by glycoside hydrolase family 10 (GH10) xylanases produces xylo- and arabinoxylo-oligosaccharides ((A)XOS) which have shown prebiotic effects. The thermostable GH10 xylanase RmXyn10A has shown great potential to produce (A)XOS. In this study, the structure of RmXyn10A was investigated, the catalytic module by homology modelling and site-directed mutagenesis and the arrangement of its five domains by small-angle X-ray scattering (SAXS). Substrate specificity was explored in silico by manual docking and molecular dynamic simulations. It has been shown in the literature that the glycone subsites of GH10 xylanases are well conserved and our results suggest that RmXyn10A is no exception. The aglycone subsites are less investigated, and the modelled structure of RmXyn10A suggests that loop ß6α6 in the aglycone part of the active site contains a non-conserved α-helix, which blocks the otherwise conserved space of subsite +2. This structural feature has only been observed for one other GH10 xylanase. In RmXyn10A, docking revealed two alternative binding regions, one on either side of the α-helix. However, only one was able to accommodate arabinose-substitutions and the mutation study suggests that the same region is responsible for binding XOS. Several non-conserved structural features are most likely to be responsible for providing affinity for arabinose-substitutions in subsites +1 and +2. The SAXS rigid model of the modular arrangement of RmXyn10A displays the catalytic module close to the cell-anchoring domain while the carbohydrate binding modules are further away, likely explaining the observed lack of contribution of the CBMs to activity.
Assuntos
Proteínas de Bactérias/genética , Endo-1,4-beta-Xilanases/química , Rhodothermus/enzimologia , Proteínas de Bactérias/química , Endo-1,4-beta-Xilanases/genética , Domínios Proteicos , Estrutura Secundária de Proteína , Rhodothermus/genéticaRESUMO
In this work, asymmetrical flow field-flow fractionation (AF4) coupled with UV/Vis, multi-angle light scattering (MALS), and differential refractive index (dRI) detectors (AF4-UV-MALS-dRI) was employed for analysis of glutamate decarboxylase (LbGadB) from Lactobacillus brevis (L. brevis). AF4 provided molecular weight (MW) (or size)-based separation of dimer, hexamer, and aggregates of LbGadB. The effect of pH on oligomerization of LbGadB was investigated, and then AF4 results were compared to those from molecular modeling. The MWs measured by AF4-UV-MALS-dRI for dimeric and hexameric forms of LbGadB were 110 and 350 kDa, respectively, which are in good agreements with those theoretically calculated (110 and 330 kDa). The molecular sizes determined by AF4-UV-MALS-dRI were also in good agreement with those obtained from molecular modeling (6 and 10 nm, respectively, for dimeric and hexameric from AF4-UV-MALS-dRI and 6.4 × 7.6 and 7.6 × 13.1 nm from molecular modeling). The effects of temperature, salt type, and salt concentration on oligomerization of LbGadB were also investigated using dynamic light scattering (DLS). It was found that the hexameric form of LbGadB was most stable at pH 6 and in presence of NaCl or KCl. The results indicate that AF4, in combination of various online detectors mentioned above, provides an effective tool for monitoring of oligomerization of LbGadB under different conditions, such as temperature, pH, type of salts, and salt concentrations.
Assuntos
Glutamato Descarboxilase/química , Levilactobacillus brevis/enzimologia , Difusão Dinâmica da Luz , Estabilidade Enzimática , Fracionamento por Campo e Fluxo , Concentração de Íons de Hidrogênio , Levilactobacillus brevis/química , Modelos Moleculares , Multimerização Proteica , Refratometria , Sais/química , TemperaturaRESUMO
Xylan has a main chain consisting of ß-1,4-linked xylose residues with diverse substituents. Endoxylanases cleave the xylan chain at cleavage sites determined by the substitution pattern and thus give different oligosaccharide product patterns. Most known endoxylanases belong to glycoside hydrolase (GH) families 10 and 11. These enzymes work well on unsubstituted xylan but accept substituents in certain subsites. The GH11 enzymes are more restricted by substituents, but on the other hand, they are normally more active than the GH10 enzymes on insoluble substrates, because of their smaller size. GH5 endoxylanases accept arabinose substituents in several subsites and require it in the - 1 subsite. This specificity makes the GH5 endoxylanases very useful for degradation of highly arabinose-substituted xylans and for the selective production of arabinoxylooligosaccharides, without formation of unsubstituted xylooligosaccharides. The GH30 endoxylanases have a related type of specificity in that they require a uronic acid substituent in the - 2 subsite, which makes them very useful for the production of uronic acid substituted oligosaccharides. The ability of dietary xylooligosaccharides to function as prebiotics in humans is governed by their substitution patterns. Endoxylanases are thus excellent tools to tailor prebiotic oligosaccharides to stimulate various types of intestinal bacteria and to cause fermentation in different parts of the gastrointestinal tract. Continuously increasing knowledge on the function of the gut microbiota and discoveries of novel endoxylanases increase the possibilities to achieve health-promoting effects.
Assuntos
Endo-1,4-beta-Xilanases/metabolismo , Glucuronatos/metabolismo , Oligossacarídeos/metabolismo , Prebióticos/microbiologia , Arabinose/metabolismo , Microbioma Gastrointestinal/fisiologia , Trato Gastrointestinal/metabolismo , Trato Gastrointestinal/microbiologia , Humanos , Especificidade por Substrato , Ácidos Urônicos/metabolismo , Xilanos/metabolismoRESUMO
The ß-glucosidase TnBgl1A catalyses hydrolysis of O-linked terminal ß-glycosidic bonds at the nonreducing end of glycosides/oligosaccharides. Enzymes with this specificity have potential in lignocellulose conversion (degrading cellobiose to glucose) and conversion of bioactive flavonoids (modification of glycosylation results in modulation of bioavailability). Previous work has shown TnBgl1A to hydrolyse 3, 4' and 7 glucosylation in flavonoids, and although conversion of 3-glucosylated substrate to aglycone was low, it was improved by mutagenesis of residue N220. To further explore structure-function relationships, the crystal structure of the nucleophile mutant TnBgl1A-E349G was determined at 1.9 Å resolution, and docking studies of flavonoid substrates were made to reveal substrate interacting residues. A series of single amino acid changes were introduced in the aglycone binding region [N220(S/F), N221(S/F), F224(I), F310(L/E), and W322(A)] of the wild type. Activity screening was made on eight glucosylated flavonoids, and kinetic parameters were monitored for the flavonoid quercetin-3-glucoside (Q3), as well as for the model substrate para-nitrophenyl-ß-d-glucopyranoside (pNPGlc). Substitution by Ser at N220 or N221 increased the catalytic efficiency on both pNPGlc and Q3. Residue W322 was proven important for substrate accomodation, as mutagenesis to W322A resulted in a large reduction of hydrolytic activity on 3-glucosylated flavonoids. Flavonoid glucoside hydrolysis was unaffected by mutations at positions 224 and 310. The mutations did not significantly affect thermal stability, and the variants kept an apparent unfolding temperature of 101°C. This work pinpoints positions in the aglycone region of TnBgl1A of importance for specificity on flavonoid-3-glucosides, improving the molecular understanding of activity in GH1 enzymes. Proteins 2017; 85:872-884. © 2016 Wiley Periodicals, Inc.
Assuntos
Aminoácidos/química , Proteínas de Bactérias/química , Isoflavonas/química , Quercetina/análogos & derivados , Thermotoga neapolitana/química , beta-Glucosidase/química , Motivos de Aminoácidos , Aminoácidos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Biocatálise , Cristalografia por Raios X , Isoflavonas/metabolismo , Cinética , Modelos Moleculares , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Quercetina/química , Quercetina/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato , Termodinâmica , Thermotoga neapolitana/enzimologia , beta-Glucosidase/genética , beta-Glucosidase/metabolismoRESUMO
In this work, we present the first XOS degrading glycoside hydrolase from Weissella, WXyn43, a two-domain enzyme from GH43. The gene was amplified from genomic DNA of the XOS utilizing Weissella strain 92, classified under the species-pair Weissella cibaria/W.confusa, and expressed in Escherichia coli. The enzyme is lacking a putative signal peptide and is, from a homology model, shown to be composed of an N-terminal 5-fold ß-propeller catalytic domain and a C-terminal ß-sandwich domain of unknown function. WXyn43 hydrolyzed short (1-4)-ß-D-xylooligosaccharides, with similar kcat/KM for xylobiose (X2) and xylotriose (X3) and clearly lower efficiency in xylotetraose (X4) conversion. WXyn43 displays the highest reported kcat for conversion of X3 (900 s(-1) at 37 °C) and X4 (770 s(-1)), and kcat for hydrolysis of X2 (907 s(-1)) is comparable with or greater than the highest previously reported. The purified enzyme adopted a homotetrameric state in solution, while a truncated form with isolated N-terminal catalytic domain adopted a mixture of oligomeric states and lacked detectable activity. The homology model shows that residues from both domains are involved in monomer-monomer hydrogen bonds, while the bonds creating dimer-dimer interactions only involved residues from the N-terminal domain. Docking of X2 and X3 in the active site shows interactions corresponding to subsites -1 and +1, while presence of a third subsite is unclear, but interactions between a loop and the reducing-end xylose of X3 may be present.
Assuntos
Proteínas de Bactérias/química , Glucuronatos/metabolismo , Oligossacarídeos/metabolismo , Weissella/enzimologia , Xilosidases/química , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Estabilidade Enzimática , Dados de Sequência Molecular , Probióticos/metabolismo , Ligação Proteica , Especificidade por Substrato , Weissella/metabolismo , Xilosidases/metabolismoRESUMO
Vegetable oils are rich in health-beneficial compounds, including fatty acids, phenolic compounds, natural antioxidants, and fat-soluble vitamins. However, oil extraction methods can influence their composition. This study aims to understand the chemical basis for developing a green process to extract oils from two Andean seeds, cañihua (Chenopodium pallidicaule) and tarwi (Lupinus mutabilis). Ethanol, considered a green solvent, is compared to petroleum ether used at the laboratory level and hexane used at the industrial scale for extracting oils. The extraction efficiency is assessed in terms of yield, fatty acids profile, polar and neutral lipids, tocopherols, phenolic compounds, and antioxidant capacity. The chemical composition of edible commercial oils, such as sunflower, rapeseed, and olive oils, was used as a reference. Hexane had the highest extraction yield, followed by petroleum ether and ethanol. However, the oils extracted with ethanol having yields of tarwi 15.5% and cañihua 5.8%, w/w showed the significatively superior content of tocopherols (α, γ, and δ); phenolic compounds; and antioxidant capacity. In addition, ethanol-extracted (EE) oils have higher levels of polar lipids, such as phosphatidylcholine and phosphatidylinositol, than those extracted with the other solvents. Remarkably, EE oils presented comparable or slightly higher levels of monounsaturated fatty acids than those extracted with hexane. Finally, compared to the commercial oils, tarwi and cañihua EE oils showed lower but acceptable levels of oleic, linoleic and palmitic acids and a wider variety of fatty acids (10 and 13, respectively). The composition of tarwi and cañahua oils extracted with ethanol includes compounds associated with nutritional and health benefits, providing a sustainable alternative for oil production.
RESUMO
Bacterial exopolysaccharides (EPS) have emerged as one of the key players in the field of heavy metal-contaminated environmental bioremediation. This study aimed to characterize and evaluate the metal biosorption potential of EPS produced by a novel Psychrobacillus strain, NEAU-3TGS, isolated from an iron ore deposit at Tamra iron mine, northern Tunisia. Genomic and pan-genomic analysis of NEAU-3TGS bacterium with nine validated published Psychrobacillus species was also performed. The results showed that the NEAU-3TGS genome (4.48 Mb) had a mean GC content of 36%, 4,243 coding sequences and 14 RNA genes. Phylogenomic analysis and calculation of nucleotide identity (ANI) values (less than 95% for new species with all strains) confirmed that NEAU-3TGS represents a potential new species. Pangenomic analysis revealed that Psychrobacillus genomic diversity represents an "open" pangenome model with 33,091 homologous genes, including 65 core, 3,738 shell, and 29,288 cloud genes. Structural EPS characterization by attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy showed uronic acid and α-1,4-glycosidic bonds as dominant components of the EPS. X-ray diffraction (XRD) analysis revealed the presence of chitin, chitosan, and calcite CaCO3 and confirmed the amorphous nature of the EPS. Heavy metal bioabsorption assessment showed that iron and lead were more adsorbed than copper and cadmium. Notably, the optimum activity was observed at 37°C, pH=7 and after 3 h contact of EPS with each metal. Genomic insights on iron acquisition and metabolism in Psychrobacillus sp. NEAU-3TGS suggested that no genes involved in siderophore biosynthesis were found, and only the gene cluster FeuABCD and trilactone hydrolase genes involved in the uptake of siderophores, iron transporter and exporter are present. Molecular modelling and docking of FeuA (protein peptidoglycan siderophore-binding protein) and siderophores ferrienterobactine [Fe+3 (ENT)]-3 and ferribacillibactine [Fe+3 (BB)]-3 ligand revealed that [Fe+3 (ENT)]-3 binds to Phe122, Lys127, Ile100, Gln314, Arg215, Arg217, and Gln252. Almost the same for [Fe+3 (ENT)]-3 in addition to Cys222 and Tyr229, but not Ile100.To the best of our knowledge, this is the first report on the characterization of EPS and the adsorption of heavy metals by Psychrobacillus species. The heavy metal removal capabilities may be advantageous for using these organisms in metal remediation.
RESUMO
Tara gum (TG) is a polysaccharide extracted from the seeds of a South American tree called Tara (Caesalpinia spinosa). TG is a galactomannan with many applications in the food industry, mainly as an emulsifier and stabilizer agent. In addition, it is also used in the paper and cosmetic industries. In the present study, we performed a molecular characterization based on chemical composition and physicochemical properties to understand the properties behind TG applications. TG was extracted and purified from Tara seeds distributed in different ecoregions of Bolivia. The monosaccharide composition analysis was determined by high-performance anion-exchange chromatography/pulsed amperometric detection (HPAEC-PAD). At the same time, their molecular characteristics, such as molar mass, root-mean-square radius, hydrodynamic radius, conformation, and densities, were studied by asymmetrical flow field-flow fractionation coupled to multi-angle light scattering refractive index (AF4-MALS-dRI), also the specific refractive index increment (dn/dc) was determined for the first time using AF4 for TG. The results revealed that the gum samples are galactomannans composed of mannose (Man) and galactose (Gal) in a ratio of 3.37 (Man/Gal), with an average molar mass range from 2.460 × 107 to 3.699 × 107 Da, distributed in a single population. The root-mean-square radius range from 260.4 to 281.6 nm, and dn/dc is 0.1454. The Kratky plots based on 14 scattering angles indicated that the conformation of all samples corresponds to random coil monodisperse, while their gyration radius/hydrodynamic radius ratio (ρ) is high. All these results suggest that the chains have a low branched density, consistent with the Gal/Man composition. To the best of our knowledge, we report for the first time an integrated physicochemical study of TG relevant to developing emulsifier and stabilizer formulations.