RESUMO
α-Glucans produced by glucansucrase enzymes hold strong potential for industrial applications. The exact determinants of the linkage specificity of glucansucrase enzymes have remained largely unknown, even with the recent elucidation of glucansucrase crystal structures. Guided by the crystal structure of glucansucrase GTF180-ΔN from Lactobacillus reuteri 180 in complex with the acceptor substrate maltose, we identified several residues (Asp-1028 and Asn-1029 from domain A, as well as Leu-938, Ala-978, and Leu-981 from domain B) near subsite +1 that may be critical for linkage specificity determination, and we investigated these by random site-directed mutagenesis. First, mutants of Ala-978 (to Leu, Pro, Phe, or Tyr) and Asp-1028 (to Tyr or Trp) with larger side chains showed reduced degrees of branching, likely due to the steric hindrance by these bulky residues. Second, Leu-938 mutants (except L938F) and Asp-1028 mutants showed altered linkage specificity, mostly with increased (α1 â 6) linkage synthesis. Third, mutation of Leu-981 and Asn-1029 significantly affected the transglycosylation reaction, indicating their essential roles in acceptor substrate binding. In conclusion, glucansucrase product specificity is determined by an interplay of domain A and B residues surrounding the acceptor substrate binding groove. Residues surrounding the +1 subsite thus are critical for activity and specificity of the GTF180 enzyme and play different roles in the enzyme functions. This study provides novel insights into the structure-function relationships of glucansucrase enzymes and clearly shows the potential of enzyme engineering to produce tailor-made α-glucans.
Assuntos
Aminoácidos/metabolismo , Glicosiltransferases/metabolismo , Limosilactobacillus reuteri/enzimologia , Sequência de Aminoácidos , Aminoácidos/química , Domínio Catalítico , Cromatografia Gasosa-Espectrometria de Massas , Glicosiltransferases/química , Glicosiltransferases/genética , Metilação , Dados de Sequência Molecular , Mutagênese , Espectroscopia de Prótons por Ressonância Magnética , Homologia de Sequência de AminoácidosRESUMO
Recently, we have shown that glycoside hydrolases enzymes of family GH17 from proteobacteria (genera Pseudomonas, Azotobacter) catalyze elongation transfer reactions with laminari-oligosaccharides generating (ß1â3) linkages preferably and to a lesser extent (ß1â6) or (ß1â4) linkages. In the present study, the cloning and characterization of the gene encoding the structurally very similar GH17 domain of the NdvB enzyme from Bradyrhizobium diazoefficiens, designated Glt20, as well as its catalytic properties are described. The Glt20 enzyme was strikingly different from the previously investigated bacterial GH17 enzymes, both regarding substrate specificity and product formation. The Azotobacter and Pseudomonas enzymes cleaved the donor laminari-oligosaccharide substrates three or four moieties from the non-reducing end, generating linear oligosaccharides. In contrast, the Glt20 enzyme cleaved donor laminari-oligosaccharide substrates two glucose moieties from the reducing end, releasing laminaribiose and transferring the remainder to laminari-oligosaccharide acceptor substrates creating only (ß1â3)(ß1â6) branching points. This enables Glt20 to transfer larger oligosaccharide chains than the other type of bacterial enzymes previously described, and helps explain the biologically significant formation of cyclic ß-glucans in B. diazoefficiens.
Assuntos
Bradyrhizobium/enzimologia , Oligossacarídeos/metabolismo , beta-Glucosidase/metabolismo , Biocatálise , Proteínas Recombinantes/metabolismo , beta-Glucosidase/genéticaRESUMO
Glucansucrases are exclusively found in lactic acid bacteria and synthesize a variety of α-glucans from sucrose. They are large multidomain enzymes belonging to the CAZy family 70 of glycoside hydrolase enzymes (GH70). The crystal structure of the N-terminal truncated GTF180 of Lactobacillus reuteri 180 (GTF180-ΔN) revealed that the polypeptide chain follows a U shape course to form five domains, including domains A, B, and C, which resemble those of family GH13 enzymes, and two extra and novel domains (domains IV and V), which are attached to the catalytic core. To elucidate the functional roles of domain V, we have deleted the domain V fragments from both the N- and C-terminal ends (GTF180-ΔNΔV). Truncation of domain V of GTF180-ΔN yielded a catalytically fully active enzyme but with heavily impaired polysaccharide synthesis ability. Instead, GTF180-ΔNΔV produced a large amount of oligosaccharides. Domain V is not involved in determining the linkage specificity, and the size of polysaccharide produced as the polysaccharide produced by GTF180-ΔNΔV was identical in size and structure with that of GTF180-ΔN. The data indicates that GTF180-ΔNΔV acts nonprocessively, frequently initiating synthesis of a new oligosaccharide from sucrose, instead of continuing the synthesis of a full size polysaccharide. Mutations L940E and L940F in GTF180-ΔNΔV, which are involved in the acceptor substrate binding, restored polysaccharide synthesis almost to the level of GTF180-ΔN. These results demonstrated that interactions of growing glucan chains with both domain V and acceptor substrate binding sites are important for polysaccharide synthesis.
Assuntos
Glicosiltransferases/metabolismo , Limosilactobacillus reuteri/enzimologia , Limosilactobacillus reuteri/metabolismo , Polissacarídeos/biossíntese , Glicosiltransferases/genética , Limosilactobacillus reuteri/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Deleção de SequênciaRESUMO
Human milk oligosaccharides (HMOs) are a major constituent of human breast milk and play an important role in reducing the risk of infections in infants. The structures of these HMOs show similarities with blood group antigens in protein glycosylation, in particular in relation to fucosylation in Lewis blood group-type epitopes, matching the maternal pattern. Previously, based on the Secretor and Lewis blood group system, four milk groups have been defined, i.e. Lewis-positive Secretors, Lewis-positive non-Secretors, Lewis-negative Secretors and Lewis-negative non-Secretors. Here, a rapid one-dimensional (1)H nuclear magnetic resonance (NMR) analysis method is presented that identifies the presence/absence of (α1-2)-, (α1-3)- and (α1-4)-linked fucose residues in HMO samples, affording the essential information to attribute different HMO samples to a specific milk group. The developed method is based on the NMR structural-reporter-group concept earlier established for glycoprotein glycans. Further evaluation of the data obtained from the analysis of 36 HMO samples shows that within each of the four milk groups the relative levels of the different fucosylation epitopes can greatly vary. The data also allow a separation of the Lewis-positive Secretor milk group into two sub-groups.
Assuntos
Epitopos/análise , Antígenos do Grupo Sanguíneo de Lewis/análise , Leite Humano/química , Oligossacarídeos/química , Epitopos/química , Epitopos/imunologia , Humanos , Antígenos do Grupo Sanguíneo de Lewis/química , Antígenos do Grupo Sanguíneo de Lewis/imunologia , Espectroscopia de Ressonância Magnética , Leite Humano/imunologia , Oligossacarídeos/análise , Oligossacarídeos/imunologia , PrótonsRESUMO
The probiotic bacterium Lactobacillus reuteri 121 produces a complex, branched (1 â 4, 1 â 6)-α-D-glucan as extracellular polysaccharide (reuteran) from sucrose (Suc), using a single glucansucrase/glucosyltransferase (GTFA) enzyme (reuteransucrase). To gain insight into the reaction/product specificity of the GTFA enzyme and the mechanism of reuteran formation, incubations with Suc and/or a series of malto-oligosaccharides (MOSs) (degree of polymerization (DP2-DP6)) were followed in time. The structures of the initially formed products, isolated via high-performance anion-exchange chromatography, were analyzed by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry and 1D/2D (1)H/(13)C NMR spectroscopy. Incubations with Suc only, acting as both donor and acceptor, resulted in elongation of Suc with glucose (Glc) units via alternating (α1 â 4) and (α1 â 6) linkages, yielding linear gluco-oligosaccharides up to at least DP ~ 12. Simultaneously with the ensemble of oligosaccharides, polymeric material was formed early on, suggesting that alternan fragments longer than DP ~ 12 have higher affinity with the GTFA enzyme and are quickly extended, yielding high-molecular-mass branched reuteran (4 × 10(7) Da). MOSs (DP2-DP6) in the absence of Suc turned out to be poor substrates. Incubations of GTFA with Suc plus MOSs as substrates resulted in preferential elongation of MOSs (acceptors) with Glc units from Suc (donor). This apparently reflects the higher affinity of GTFA for MOSs compared with Suc. In accordance with the GTFA specificity, most prominent products were oligosaccharides with an (α1 â 4)/(α1 â 6) alternating structure.
Assuntos
Glicosiltransferases/metabolismo , Limosilactobacillus reuteri/enzimologia , Oligossacarídeos/metabolismo , Sacarose/metabolismoRESUMO
The glycopeptide CcTx, isolated from the venom of the piscivorous cone snail Conus consors, belongs to the κA-family of conopeptides. These toxins elicit excitotoxic responses in the prey by acting on voltage-gated sodium channels. The structure of CcTx, a first in the κA-family, has been determined by high-resolution NMR spectroscopy together with the analysis of its O-glycan at Ser7. A new type of glycopeptide O-glycan core structure, here registered as core typeâ 9, containing two terminal L-galactose units {α-L-Galp-(1â4)-α-D-GlcpNAc-(1â6)-[α-L-Galp-(1â2)-ß-D-Galp-(1â3)-]α-D-GalpNAc-(1âO)}, is highlighted. A sequence comparison to other putative members of the κA-family suggests that O-linked glycosylation might be more common than previously thought. This observation alone underlines the requirement for more careful and in-depth investigations into this type of post-translational modification in conotoxins.
Assuntos
Caramujo Conus/química , Glicopeptídeos/química , Venenos de Moluscos/química , Animais , Glicosilação , Espectroscopia de Ressonância Magnética , Estrutura MolecularRESUMO
Conotoxins are small peptides present in the venom of cone snails. The snail uses this venom to paralyze and capture prey. The constituent conopeptides display a high level of chemical diversity and are of particular interest for scientists as tools employed in neurological studies and for drug development, because they target with exquisite specificity membrane receptors, transporters, and various ion channels in the nervous system. However, these peptides are known to contain a high frequency and variability of post-translational modifications-including sometimes O-glycosylation-which are of importance for biological activity. The potential application of specific conotoxins as neuropharmalogical agents and chemical probes requires a full characterization of the relevant peptides, including the structure of the carbohydrate part. In this review, the currently existing knowledge of O-glycosylation of conotoxins is described.
Assuntos
Conotoxinas/química , Caramujo Conus/química , Desenho de Fármacos , Animais , Conotoxinas/isolamento & purificação , Conotoxinas/farmacologia , Glicosilação , Humanos , Terapia de Alvo Molecular , Processamento de Proteína Pós-TraducionalRESUMO
Recently, a novel glucansucrase (GS)-like gene (gtfB) was isolated from the probiotic bacterium Lactobacillus reuteri 121 and expressed in Escherichia coli. The purified recombinant GTFB enzyme was characterized and turned out to be inactive with sucrose, the natural GS substrate. Instead, GTFB acted on malto-oligosaccharides (MOSs), thereby yielding elongated gluco-oligomers/polymers containing besides (α1 â 4) also (α1 â 6) glycosidic linkages, and it was classified as a 4,6-α-glucanotransferase. To gain more insight into its reaction specificity, incubations of the GTFB enzyme with a series of MOSs and their corresponding alditols [degree of polymerization, DP2(-ol)-DP7(-ol)] were carried out, and (purified) products were structurally analyzed with matrix-assisted laser desorption ionization time-of-flight mass spectrometry and one-/two-dimensional (1)H and (13)C nuclear magnetic resonance spectroscopy. With each of the tested malto-oligomers, the GTFB enzyme yielded series of novel linear isomalto-/malto-oligomers, in the case of DP7 up to DP >35.
Assuntos
Proteínas de Bactérias/química , Glucosiltransferases/química , Limosilactobacillus reuteri/enzimologia , Maltose/química , Oligossacarídeos/síntese química , Configuração de Carboidratos , Sequência de Carboidratos , Cromatografia por Troca Iônica , Glucanos/química , Glicosilação , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Oligossacarídeos/química , Oligossacarídeos/isolamento & purificação , Proteínas Recombinantes/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Álcoois Açúcares/química , Trissacarídeos/químicaRESUMO
Hemagglutinin esterases (HEs), closely related envelope glycoproteins in influenza C and corona- and toroviruses, mediate reversible attachment to O-acetylated sialic acids (Sias). They do so by acting both as lectins and as receptor-destroying enzymes, functions exerted by separate protein domains. HE divergence was accompanied by changes in quaternary structure and in receptor and substrate specificity. The selective forces underlying HE diversity and the molecular basis for Sia specificity are poorly understood. Here we present crystal structures of porcine and bovine torovirus HEs in complex with receptor analogs. Torovirus HEs form homodimers with sialate-O-acetylesterase domains almost identical to corresponding domains in orthomyxo- and coronavirus HEs, but with unique lectin sites. Structure-guided biochemical analysis of the esterase domains revealed that a functionally, but not structurally conserved arginine-Sia carboxylate interaction is critical for the binding and positioning of glycosidically bound Sias in the catalytic pocket. Although essential for efficient de-O-acetylation of Sias, this interaction is not required for catalysis nor does it affect substrate specificity. In fact, the distinct preference of the porcine torovirus enzyme for 9-mono- over 7,9-di-O-acetylated Sias can be explained from a single-residue difference with HEs of more promiscuous specificity. Apparently, esterase and lectin pockets coevolved; also the porcine torovirus HE receptor-binding site seems to have been designed to use 9-mono- and exclude di-O-acetylated Sias, possibly as an adaptation to replication in swine. Our findings shed light on HE evolution and provide fundamental insight into mechanisms of substrate binding, substrate recognition, and receptor selection in this important class of virion proteins.
Assuntos
Hemaglutininas Virais/química , Hemaglutininas Virais/metabolismo , Torovirus/enzimologia , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/metabolismo , Substituição de Aminoácidos , Animais , Domínio Catalítico/genética , Bovinos , Cristalografia por Raios X , Dimerização , Hemaglutininas Virais/genética , Ligantes , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ácidos Siálicos/metabolismo , Especificidade da Espécie , Especificidade por Substrato , Suínos , Torovirus/genética , Proteínas Virais de Fusão/genéticaRESUMO
Alginate (alginic acid) is a linear polysaccharide, wherein (1â4)-linked ß-D-mannuronic acid and its C5 epimer, α-L-guluronic acid, are arranged in varying sequences. Alginate lyases catalyze the depolymerization of alginate, thereby cleaving the (1â4) glycosidic linkages between the monomers by a ß-elimination mechanism, to yield unsaturated 4-deoxy-L-erythro-hex-4-enopyranosyluronic acid (Δ) at the non-reducing end of resulting oligosaccharides (α-L-erythro configuration) or, depending on the enzyme, the unsaturated monosaccharide itself. In solution, the released free unsaturated monomer product is further hydrated in a spontaneous (keto-enol tautomerization) process to form two cyclic stereoisomers. In this study, two alginate lyase genes, designated alyRm3 and alyRm4, from the marine thermophilic bacterium Rhodothermus marinus (strain MAT378), were cloned and expressed in Escherichia coli. The recombinant enzymes were characterized, and their substrate specificity and product structures determined. AlyRm3 (PL39) and AlyRm4 (PL17) are among the most thermophilic and thermostable alginate lyases described to date with temperature optimum of activity at â¼75 and 81°C, respectively. The pH optimum of activity of AlyRm3 is â¼5.5 and AlyRm4 at pH 6.5. Detailed NMR analysis of the incubation products demonstrated that AlyRm3 is an endolytic lyase, while AlyRm4 is an exolytic lyase, cleaving monomers from the non-reducing end of oligo/poly-alginates.
RESUMO
Over the years several ß-glucan transferases from yeast and fungi have been reported, but enzymes with such an activity from bacteria have not been characterized so far. In this work, we describe the cloning and expression of genes encoding ß-glucosyltransferase domains of glycosyl hydrolase family GH17 from three species of proteobacteria: Pseudomonas aeruginosa PAO1, P. putida KT2440 and Azotobacter vinelandii ATCC BAA-1303. The encoded enzymes of these GH17 domains turned out to have a non-Leloir trans-ß-glucosylation activity, as they do not use activated nucleotide sugar as donor, but transfer a glycosyl group from a ß-glucan donor to a ß-glucan acceptor. More particularly, the activity of the three recombinant enzymes on linear (ß1 â 3)-linked gluco-oligosaccharides (Lam-Glc(4-9)) and their corresponding alditols (Lam-Glc(4-9)-ol) was studied. Detailed structural analysis, based on thin-layer chromatography, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, electrospray ionization mass spectrometry, and 1D/2D (1)H and (13)C nuclear magnetic resonance data, revealed diverse product spectra. Depending on the enzyme used, besides (ß1 â 3)-elongation activity, (ß1 â 4)- or (ß1 â 6)-elongation, or (ß1 â 6)-branching activities were also detected.
Assuntos
Azotobacter vinelandii/enzimologia , Glucosiltransferases/biossíntese , Polissacarídeos/metabolismo , Pseudomonas aeruginosa/enzimologia , Pseudomonas putida/enzimologia , Ensaios Enzimáticos , Glucanos , Glucosiltransferases/química , Modelos Moleculares , Estrutura Molecular , Conformação Proteica , beta-Glucanas/químicaRESUMO
Starch-acting α-glucanotransferase enzymes are of great interest for applications in the food industry. In previous work, we have characterized various 4,6- and 4,3-α-glucanotransferases of the glycosyl hydrolase (GH) family 70 (subfamily GtfB), synthesizing linear or branched α-glucans. Thus far, GtfB enzymes have only been identified in mesophilic Lactobacilli. Database searches showed that related GtfC enzymes occur in Gram-positive bacteria of the genera Exiguobacterium, Bacillus, and Geobacillus, adapted to growth at more extreme temperatures. Here, we report characteristics of the Geobacillus sp. 12AMOR1 GtfC enzyme, with an optimal reaction temperature of 60 °C and a melting temperature of 68 °C, allowing starch conversions at relatively high temperatures. This thermostable 4,6-α-glucanotransferase has a novel product specificity, cleaving off predominantly maltose units from amylose, attaching them with an (α1 â 6)-linkage to acceptor substrates. In fact, this GtfC represents a novel maltogenic α-amylase. Detailed structural characterization of its starch-derived α-glucan products revealed that it yielded a unique polymer with alternating (α1 â 6)/(α1 â 4)-linked glucose units but without branches. Notably, this Geobacillus sp. 12AMOR1 GtfC enzyme showed clear antistaling effects in bread bakery products.
Assuntos
Geobacillus , Sistema da Enzima Desramificadora do Glicogênio , Pão , Geobacillus/genética , Glucanos , Sistema da Enzima Desramificadora do Glicogênio/genéticaRESUMO
The presence of different derivatives of sialic acids (SA) on Leishmania donovani instigated us to investigate their status on different strains of Leishmania sp. causing different forms of the disease. Leishmania tropica (K27), Leishmania major (JISH118) and Leishmania mexicana (LV4) responsible for cutaneous, Leishmania braziliensis (L280) and Leishmania amazonensis (LV81) causing diffuse and Leishmania infantum (MON29) responsible for visceral leishmaniasis were included in this study. The strains showed a differential distribution of SA in spite of their close resemblance in pathogenesis. K27, JISH118, L280 and MON29 were categorized as high SA-containing strains having enhanced 9-O-acetyl sialic acid (9-O-AcSA(high)) whereas LV4 and LV81 evidenced considerably reduced SA. Interestingly, 9-O-AcSA(high) promastigotes showed significant viability as compared to their de-O-acetylated forms after exposure to NaNO(2) suggesting the involvement of 9-O-AcSA in conferring nitric oxide (NO) resistance. Enhanced intracellular survivability was demonstrated following infection of human macrophages with 9-O-AcSA(high) promastigotes in contrast to their de-O-acetylated forms indicating their contribution in bestowing a survival benefit. Additionally, reduced accumulation of NO, interleukin-12 and interferon-gamma in the supernatant of macrophages infected with 9-O-AcSA(high) promastigotes indicated suppression of leishmanicidal host responses. However, LV4 and LV81 with least 9-O-AcSA, before and after de-O-acetylation, showed unaltered NO resistance, multiplicity and host responses signifying the probable involvement of other determinants which may be a function of their inherent parasitic attribute. Hence, enhanced levels of 9-O-AcSA serve as one of the potential determinants responsible for increased NO resistance and survivability of parasites by inhibition of host responses.
Assuntos
Leishmania/química , Leishmania/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/parasitologia , Óxido Nítrico/antagonistas & inibidores , Ácidos Siálicos/farmacologia , Células Cultivadas , Humanos , Leishmania/imunologia , Leishmaniose/imunologia , Leishmaniose/parasitologia , Macrófagos/imunologia , Macrófagos/metabolismo , Óxido Nítrico/metabolismo , Testes de Sensibilidade Parasitária , Ácidos Siálicos/química , Ácidos Siálicos/imunologia , Fatores de TempoRESUMO
The human pathogen Cryptococcus neoformans causes meningoencephalitis. The polysaccharide capsule is one of the main virulence factors and consists of two distinct polysaccharides, glucuronoxylomannan (GXM) and galactoxylomannan (GalXM). How capsular polysaccharides are synthesized, transported, and assembled is largely unknown. Previously, it was shown that mutations in the CAP10, CAP59, CAP60, and CAP64 genes result in an acapsular phenotype. Here, it is shown that these acapsular mutants do secrete GalXM and GXM-like polymers. GXM and GalXM antibodies specifically reacted with whole cells and the growth medium of the wild type and CAP mutants, indicating that the capsule polysaccharides adhere to the cell wall and are shed into the environment. These polysaccharides were purified from the medium, either with or without anion-exchange chromatography. Monosaccharide analysis of polysaccharide fractions by gas-liquid chromatography/mass spectrometry showed that wild-type cells secrete both GalXM and GXM. The CAP mutants, on the other hand, were shown to secrete GalXM and GXM-like polymers. Notably, the GalXM polymers were shown to contain glucuronic acid. One-dimensional (1)H nuclear magnetic resonance confirmed that the CAP mutants secrete GalXM and also showed the presence of O-acetylated polymers. This is the first time it is shown that CAP mutants secrete GXM-like polymers in addition to GalXM. The small amount of this GXM-like polymer, 1 to 5% of the total amount of secreted polysaccharides, may explain the acapsular phenotype.
Assuntos
Cryptococcus neoformans/genética , Cryptococcus neoformans/metabolismo , Espaço Extracelular/metabolismo , Proteínas Fúngicas/genética , Mutação , Polissacarídeos/metabolismo , Transporte Biológico , Cryptococcus neoformans/química , Espaço Extracelular/química , Proteínas Fúngicas/metabolismo , Polissacarídeos/química , Polissacarídeos/isolamento & purificaçãoRESUMO
Mutagenesis of specific amino acid residues of the glucansucrase (GTF180) enzyme from Lactobacillus reuteri strain 180 yielded 12 mutant enzymes that produced modified exopolysaccharides (mEPSs) from sucrose. Ethanol-precipitated and purified mEPSs were subjected to linkage analysis, Smith degradation analysis, and 1D/2D (1)H NMR spectroscopy. Comparison of the results with structural data of the previously described wild type EPS180 and triple mutant mEPS-PNNS revealed a broad variation of structural elements between mEPS molecules. The amount of (alpha1-->3) linkages varied from 14-43%, the amount of (alpha1-->4) linkages (not present in the wild type) from 0-12%, and the amount of (alpha1-->6) linkages from 51-86%. The average molecular weight (M(w)) ranged from 9.4 to 32.3 MDa and the degree of branching varied from 8-20%. Using a previously established (1)H NMR structural-reporter-group concept, composite models, that include all identified structural features, were formulated for all mEPS molecules. Variations in the mEPS structures strongly affected the physical properties of the mEPSs.
Assuntos
Glicosiltransferases/metabolismo , Limosilactobacillus reuteri/enzimologia , Polissacarídeos Bacterianos/metabolismo , Engenharia de Proteínas , Substituição de Aminoácidos , Biocatálise , Configuração de Carboidratos , Glicosiltransferases/química , Glicosiltransferases/genética , Mutagênese Sítio-Dirigida , Mutação , Polissacarídeos Bacterianos/química , Sacarose/químicaRESUMO
Production of exopolysaccharides by lactic acid bacteria is a common phenomenon. Structural information of these widely diverse biopolymers is rendered by the monosaccharide composition, the anomeric configurations, the type of glycosidic linkages, the presence of repeating units and noncarbohydrate substituents, and finally the presentation of a chemical molecular structure or composite model. The detailed structural analysis of polysaccharides is a time-consuming pursuit, including the use of different techniques, such as chemical degradation methods (e.g., hydrolysis), separation methods (e.g., SEC-chromatography and HPLC/HPAEC), and identification methods (e.g., GLC-EIMS and 1H/13C NMR spectroscopy). In this chapter, some analytical methods are described and demonstrated for two different exopolysaccharides from lactic acid bacteria.
Assuntos
Lactobacillales/química , Estrutura Molecular , Polissacarídeos Bacterianos/química , Acetilação , Glicosídeos , Hidrólise , Lactobacillales/metabolismo , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Monossacarídeos/química , Oxirredução , Polissacarídeos Bacterianos/metabolismoRESUMO
The adverse health effects of sucrose overconsumption, typical for diets in developed countries, necessitate use of low-calorie sweeteners. Following approval by the European Commission (2011), steviol glycosides are increasingly used as high-intensity sweeteners in food. Stevioside is the most prevalent steviol glycoside in Stevia rebaudiana plant leaves, but it has found limited applications in food products due to its lingering bitterness. Enzymatic glucosylation is a strategy to reduce stevioside bitterness, but reported glucosylation reactions suffer from low productivities. Here we present the optimized and efficient α-glucosylation of stevioside using the mutant glucansucrase Gtf180-ΔN-Q1140E and sucrose as donor substrate. Structures of novel products were elucidated by NMR spectroscopy, mass spectrometry and methylation analysis; stevioside was mainly glucosylated at the steviol C-19 glucosyl moiety. Sensory analysis of the α-glucosylated stevioside products by a trained panel revealed a significant reduction in bitterness compared to stevioside, resulting in significant improvement of edulcorant/organoleptic properties.
Assuntos
Proteínas de Bactérias/metabolismo , Diterpenos do Tipo Caurano/metabolismo , Glucosídeos/metabolismo , Glicosiltransferases/metabolismo , Paladar/fisiologia , Proteínas de Bactérias/genética , Diterpenos do Tipo Caurano/análise , Glucosídeos/análise , Glicosilação , Glicosiltransferases/genética , Humanos , Isomerismo , Lactobacillus/enzimologia , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Mutagênese , Folhas de Planta/metabolismo , Stevia/metabolismo , Sacarose/química , Sacarose/metabolismo , Edulcorantes/análise , Edulcorantes/metabolismoRESUMO
Site-directed mutagenesis of the glucansucrase gtf180 gene from Lactobacillus reuteri strain 180 was used to transform the active site region. The alpha-D-glucan ( mEPS-PNNS) produced by the triple mutant V1027P:S1137N:A1139S differed in structure from that of the wild-type alpha-D-glucan ( EPS180). Besides (alpha1-->3) and (alpha1-->6) linkages, as present in EPS180, mEPS-PNNS also contained (alpha1-->4) linkages. Linkage analysis, periodate oxidation, and 1D/2D (1)H NMR spectroscopy of the intact mEPS-PNNS, as well as MS and NMR analysis of oligosaccharides obtained by partial acid hydrolysis of mEPS-PNNS afforded a composite model, which includes all identified structural features.
Assuntos
Glicosiltransferases/química , Glicosiltransferases/genética , Limosilactobacillus reuteri/enzimologia , Limosilactobacillus reuteri/genética , Mutação , Ânions , Cromatografia por Troca Iônica/métodos , Glucanos , Concentração de Íons de Hidrogênio , Hidrólise , Ácido Láctico/metabolismo , Espectroscopia de Ressonância Magnética , Espectrometria de Massas/métodos , Mutagênese Sítio-Dirigida , Oxigênio/química , Ácido Periódico/química , Engenharia de Proteínas/métodosRESUMO
The exopolysaccharides produced by three propionibacteria strains, Propionibacterium freudenreichii 109, Propionibacterium freudenreichii 111, and Propionibacterium thoenii 126, grown on whey-based media, were found to be charged heteropolymers, composed of D-glucose, D-mannose, and D-glucuronic acid in molar ratios of 2:2:1. By means of methylation analysis, mass spectrometry, partial acid hydrolysis, and 1D/2D NMR (1H and 13C) studies, it was determined that all three exopolysaccharides contain the same branched, pentasaccharide repeating unit: [Formula: see text].
Assuntos
Polissacarídeos Bacterianos/química , Propionibacterium/química , Ácidos/química , Sequência de Carboidratos , Hidrólise , Espectroscopia de Ressonância Magnética , Metilação , Dados de Sequência Molecular , Polissacarídeos Bacterianos/biossíntese , Polissacarídeos Bacterianos/isolamento & purificação , Propionibacterium/metabolismoRESUMO
An NMR study of proton chemical shift patterns of known linear alpha-D-glucopyranose di- and trisaccharide structures was carried out. Chemical shift patterns for (alpha1-->2)-, (alpha1-->3)-, (alpha1-->4)- and (alpha1-->6)-linked D-glucose residues were analysed and compared to literature data. Using these data, a 1H NMR structural-reporter-group concept was formulated to function as a tool in the structural analysis of alpha-D-glucans.