RESUMEN
Peanuts are one of the most common and severe food allergens. Nevertheless, the occurrence of peanut allergy varies between countries and depends on both the exposure and the way peanuts are consumed. Processing is known to influence the allergenicity of peanut proteins. The aim of this study was to assess the effect of thermal processing on the IgE-binding capacity of whole peanut protein extracts and of the major peanut allergens Ara h 1 and Ara h 2. Whole proteins, Ara h 1, and Ara h 2 were extracted and purified from raw, roasted and boiled peanuts using selective precipitation and multiple chromatographic steps, and were then characterized by electrophoresis and mass spectrometry. The immunoreactivity of whole peanut extracts and purified proteins was analyzed by the enzyme allergosorbent test (EAST) and EAST inhibition using the sera of 37 peanut-allergic patients. The composition of the whole protein extracts was modified after heat processing, especially after boiling. The electrophoretic pattern showed protein bands of low molecular weight that were less marked in boiled than in raw and roasted peanuts. The same low-molecular-weight proteins were found in the cooking water of peanuts. Whole peanut protein extracts obtained after the different processes were all recognized by the IgE of the 37 patients. The IgE-binding capacity of the whole peanut protein extracts prepared from boiled peanuts was 2-fold lower than that of the extracts prepared from raw and roasted peanuts. No significant difference was observed between protein extracts from raw and roasted peanuts. It is noteworthy that the proteins present in the cooking water were also recognized by the IgE of peanut-allergic patients. IgE immunoreactivity of purified Ara h 1 and Ara h 2 prepared from roasted peanuts was higher than that of their counterparts prepared from raw and boiled peanuts. The IgE-binding capacity of purified Ara h 1 and Ara h 2 was altered by heat treatment and in particular was increased by roasting. However, no significant difference in IgE immunoreactivity was observed between whole protein extracts from raw and roasted peanuts. The decrease in allergenicity of boiled peanuts results mainly from a transfer of low-molecular-weight allergens into the water during cooking.
Asunto(s)
Alérgenos/química , Alérgenos/inmunología , Arachis/química , Calor , Proteínas de Plantas/química , Proteínas de Plantas/inmunología , Albuminas 2S de Plantas , Antígenos de Plantas , Manipulación de Alimentos/métodos , Glicoproteínas/química , Glicoproteínas/inmunología , Humanos , Inmunoglobulina E/metabolismo , Proteínas de la Membrana , Hipersensibilidad al Cacahuete/inmunología , Extractos Vegetales/inmunologíaRESUMEN
The immobilization of dextransucrase in Ca-alginate beads relies on the close association between dextran polymer and dextransucrase. However, high amounts of dextran in the enzyme preparation drastically limit the specific activity of the immobilized enzyme (4 U/mL of alginate beads). Moreover, even in the absence of diffusion limitation at the batch conditions used, the enzyme behavior is modified by entrapment so that the dextran yield increases and the alpha-1,2 glucooligosaccharides (GOS) are produced with a lower yield (46.6% instead of 56.7%) and have a lower mean degree of polymerization than with the free dextransucrase. When the immobilized catalyst is used in a continuous reaction, the reactor flow rate necessary to obtain high conversion of the substrates is very low, leading to external diffusion resistance. As a result, dextran synthesis is even higher than in the batch reaction, and its accumulation within the alginate beads limits the operational stability of the catalyst and decreases glucooligosaccharide yield and productivity. This effect can be limited by using reactor columns with length to diameter ratio > or =20, and by optimizing the substrate concentrations in the feed solution: the best productivity obtained was 3.74 g. U(-1). h(-1), with an alpha-1,2 GOS yield of 36%.
Asunto(s)
Reactores Biológicos , Enzimas Inmovilizadas/metabolismo , Glucosiltransferasas/metabolismo , Leuconostoc/enzimología , Oligosacáridos/biosíntesis , Cromatografía Líquida de Alta Presión , Leuconostoc/metabolismoRESUMEN
The optimization of alpha-1,2 glucooligosaccharide (GOS) synthesis from maltose and sucrose by Leuconostoc mesenteroides NRRL B-1299 dextransucrase was achieved using experimental design and consecutive analysis of the key parameters. An increase of the pH of the reaction from 5.4 to 6.7 and of the temperature from 25 to 40 degrees C significantly favored alpha-1,2 GOS synthesis, thanks to a significant decrease of the side reactions, i.e., dextran and leucrose synthesis. These positive effects were not sufficient to compensate for the decrease of enzyme stability caused by the use of high pH and temperature. However, the critical parameters were the sucrose to maltose concentration ratio (S/M) and the total sugar concentration (TSC). Alpha1,2 GOS synthesis was favored at high S/M ratios. But using these conditions also led to an increase of side reactions which could be modulated by choosing the appropriate TSC. Finally, with S/M = 4 and TSC = 45% w/v, dextran and leucrose productions were limited and the final alpha-1,2 GOS yield reached 56.7%, the total GOS yield being 88%.
Asunto(s)
Glucosa/química , Glucosa/metabolismo , Glucosiltransferasas/biosíntesis , Glucosiltransferasas/química , Leuconostoc/enzimología , Oligosacáridos/biosíntesis , Oligosacáridos/química , Cromatografía Líquida de Alta Presión , Dextranos/química , Disacáridos/química , Modelos Químicos , Modelos Estadísticos , Sensibilidad y EspecificidadRESUMEN
Cellobiose was tested as acceptor in the reaction catalyzed by alternansucrase (EC 2.4.1.140) from Leuconostoc mesenteroides NRRL B-23192. The oligosaccharides synthesized were compared to those obtained with dextransucrase from L. mesenteroides NRRL B-512F. With alternansucrase and dextransucrase, overall oligosaccharide synthesis yield reached 30 and 14%, respectively, showing that alternansucrase is more efficient than dextransucrase for cellobiose glucosylation. Interestingly, alternansucrase produced a series of oligosaccharides from cellobiose. Their structure was determined by mass spectrometry and [13C-1H] NMR spectroscopy. Two trisaccharides are first produced: alpha-D-glucopyranosyl-(1-->2)-[beta-D-glucopyranosyl-(1-->4)]-D-glucopyranose (compound A) and alpha-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl-(1-->4)-D-glucopyranose (compound B). Then, compound B can in turn be glucosylated leading to the synthesis of a tetrasaccharide with an additional alpha-(1-->6) linkage at the non-reducing end (compound D). The presence of the alpha-(1-->3) linkage occurred only in the pentasaccharides (compounds C1 and C2) formed from tetrasaccharide D. Compounds B, C1, C2 and D were never described before. They were produced efficiently only by alternansucrase. Their presence emphasizes the difference existing in the acceptor reaction selectivity of the various glucansucrases.
Asunto(s)
Celobiosa/metabolismo , Glicósido Hidrolasas/metabolismo , Glicosiltransferasas , Oligosacáridos/biosíntesis , Sacarosa/metabolismo , Secuencia de Carbohidratos , Glucosiltransferasas/metabolismo , Leuconostoc/enzimología , Datos de Secuencia Molecular , Resonancia Magnética Nuclear BiomolecularRESUMEN
Amylosucrase (E.C. 2.4.1.4) is a member of Family 13 of the glycoside hydrolases (the alpha-amylases), although its biological function is the synthesis of amylose-like polymers from sucrose. The structure of amylosucrase from Neisseria polysaccharea is divided into five domains: an all helical N-terminal domain that is not similar to any known fold, a (beta/alpha)(8)-barrel A-domain, B- and B'-domains displaying alpha/beta-structure, and a C-terminal eight-stranded beta-sheet domain. In contrast to other Family 13 hydrolases that have the active site in the bottom of a large cleft, the active site of amylosucrase is at the bottom of a pocket at the molecular surface. A substrate binding site resembling the amylase 2 subsite is not found in amylosucrase. The site is blocked by a salt bridge between residues in the second and eight loops of the (beta/alpha)(8)-barrel. The result is an exo-acting enzyme. Loop 7 in the amylosucrase barrel is prolonged compared with the loop structure found in other hydrolases, and this insertion (forming domain B') is suggested to be important for the polymer synthase activity of the enzyme. The topology of the B'-domain creates an active site entrance with several ravines in the molecular surface that could be used specifically by the substrates/products (sucrose, glucan polymer, and fructose) that have to get in and out of the active site pocket.
Asunto(s)
Glucosiltransferasas/metabolismo , alfa-Amilasas/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Glucosiltransferasas/química , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , Pliegue de ProteínaRESUMEN
The enzymatic synthesis of a mixture of unsaturated fatty acid alpha-butylglucoside esters, containing more than 60% alpha-butylglucoside linoleate, was achieved through lipase-catalyzed esterification. The continuous evaporation under reduced pressure of the water produced enabled substrate conversions greater than 95% to be reached. Two immobilized lipases from Candida antarctica (Chirazyme L2, c.-f., C2) and Rhizomucor miehei (Chirazyme L9, c.-f.) were compared in stirred batch and packed bed configurations. When the synthesis was carried out in stirred batch mode, C. antarctica lipase appeared to be of greater interest than the R. miehei enzyme in terms of stability and regioselectivity. Surprisingly, a change in the process design to a packed bed configuration enabled the stability of R. miehei lipase to be significantly improved, while the C. antarctica lipase efficiency to synthesize unsaturated fatty acid alpha-butylglucoside esters was slightly decreased. Water content in the microenvironment of the biocatalyst was assumed to be responsible for such changes. When the process is run in stirred batch mode, the conditions used promote the evaporation of the essential water surrounding the enzyme, which probably leads to R. miehei lipase dehydration. In contrast, the packed bed design enabled such water evaporation in the microenvironment of the biocatalyt to be avoided, which resulted in a tremendous improvement of R. miehei lipase stability. However, C. antarctica lipase led to the formation of 3% diesters, whereas the final percentage of diesters reached 21% when R. miehei enzyme was used as biocatalyst. A low content of diesters is of greater interest in terms of alpha-butylglucoside linoleate application as linoleic acid carrier, and therefore the enzyme choice will have to be made depending on the properties expected for the final product.
Asunto(s)
Glucósidos/síntesis química , Ácidos Linoleicos/síntesis química , Reactores Biológicos , Cromatografía en Capa Delgada , Enzimas Inmovilizadas/química , Lipasa/química , Proyectos PilotoRESUMEN
Amylosucrase produces an insoluble alpha-1,4-linked glucan from sucrose, releasing fructose. In addition to polymerisation, in the presence of sucrose as sole substrate, amylosucrase catalyses sucrose hydrolysis and oligosaccharide synthesis in significant proportions. The effects of both glycogen acceptor and sucrose concentrations on the reactions catalysed by the highly purified amylosucrase from Neisseria polysaccharea were investigated. Sucrose hydrolysis decreased strongly with the increase of the concentration of glycogen, as did oligosaccharide synthesis, by glucose transfer onto glucose and fructose. The glucosyl units consumed were then preferentially used for elongation of glycogen chains. The study of the kinetic behaviour of amylosucrase revealed a strong, sucrose concentration dependent activator effect of glycogen. This activation was decreased at high sucrose concentration. The optimal sucrose concentrations increased with glycogen concentration, suggesting competition between sucrose and glycogen, and the presence of a second non-catalytic acceptor binding site which could bind various acceptors (glucose, maltose, glycogen) and also sucrose.
Asunto(s)
Glucosiltransferasas/metabolismo , Glucógeno/metabolismo , Neisseria/enzimología , Activación Enzimática , Glucosiltransferasas/genética , Cinética , Neisseria/genética , Proteínas Recombinantes de Fusión/metabolismo , Sacarosa/metabolismoRESUMEN
Amylosucrase is a glucosyltransferase that synthesises an insoluble alpha-glucan from sucrose. The catalytic properties of the highly purified amylosucrase from Neisseria polysaccharea were characterised. Contrary to previously published results, it was demonstrated that in the presence of sucrose alone, several reactions are catalysed, in addition to polymer synthesis: sucrose hydrolysis, maltose and maltotriose synthesis by successive transfers of the glucosyl moiety of sucrose onto the released glucose, and finally turanose and trehalulose synthesis - these two sucrose isomers being obtained by glucosyl transfer onto fructose. The effect of initial sucrose concentration on initial activity demonstrated a non-Michaelian profile never previously described.
Asunto(s)
Glucosiltransferasas/metabolismo , Neisseria/enzimología , Sacarosa/metabolismo , Catálisis/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Disacáridos/metabolismo , Relación Dosis-Respuesta a Droga , Fructosa/metabolismo , Fructosa/farmacología , Glucosa/metabolismo , Glucosiltransferasas/aislamiento & purificación , Hidrólisis/efectos de los fármacos , Isomerismo , Cinética , Espectroscopía de Resonancia Magnética , Maltosa/metabolismo , Polímeros/química , Polímeros/metabolismo , Solubilidad , Sacarosa/química , Sacarosa/farmacología , Trisacáridos/metabolismoRESUMEN
Recombinant amylosucrase from Neisseria polysaccharea was crystallized by the vapour-diffusion procedure in the presence of polyethylene glycol 6000. The crystals belong to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 95.7, b = 117.2, c = 62.1 A, and diffract to 1.6 A resolution. A p-chloromercuribenzene sulfonate (pcmbs) derivative has been identified and a selenomethionine-substituted protein has been produced and crystallized.
Asunto(s)
Glucosiltransferasas/química , Neisseria/enzimología , Proteínas Recombinantes/química , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Dicroismo Circular , Cristalización , Cristalografía por Rayos X , Escherichia coli/química , Escherichia coli/enzimología , Glucosiltransferasas/biosíntesis , Glucosiltransferasas/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Unsaturated fatty acid alpha-butylglucoside esters were prepared by enzymatic esterification of alpha-butylglucoside in nonaqueous media. Conditions were firstly optimized using oleic acid as acyl group. Synthesis was possible in several solvents but the presence of water co-product in the medium limited the reaction to a thermodynamic equilibrium corresponding to a maximal conversion yield of 62%. In pure molten substrates, the removal of water under reduced pressure enabled yields superior to 95% to be obtained. Product profiles depended on enzyme origin : whatever the support, immobilized lipase B from Candida antarctica proved to be far more regioselective for the primary hydroxyl group of glucose than immobilized lipase from Rhizomucor miehei. Results obtained could be easily transposed to the acylation of alpha-butylglucoside with a commercial mixture of unsaturated fatty acids containing more than 60% of linoleic acid. The biocatalyst could be recycled more than ten times without any significant activity loss.
Asunto(s)
Cosméticos/química , Cosméticos/síntesis química , Ácidos Grasos Insaturados/síntesis química , Glucósidos/síntesis química , Ácidos Linoleicos/síntesis química , Lipasa/química , Pentanoles , Acilación , Butanoles/síntesis química , Estabilidad de Enzimas , Ésteres/síntesis química , Ésteres/química , Ácido Linoleico/química , Espectroscopía de Resonancia Magnética , Espectrometría de Masas/métodos , Ácido Oléico/química , Solventes/química , Ácido alfa-Linolénico/químicaRESUMEN
Glucansucrases are produced principally by Leuconostoc mesenteroides and oral Streptococcus species, but also by the lactic acid bacteria (Lactococci, Lactobacilli). They catalyse the synthesis of high molecular weight D-glucose polymers, named glucans, from sucrose. In the presence of efficient acceptors, they catalyse the synthesis of low molecular weight oligosaccharides. Glucosidic bond synthesis occurs without the mediation of nucleotide activated sugars and cofactors are not necessary. Glucansucrases have an industrial value because of the production of dextrans and oligosaccharides and a biological importance by their key role in the cariogenic process. They were identified more than 50 years ago. The first glucansucrase encoding gene was cloned more than 10 years ago. But the mechanism of their action remains incompletely understood. However, in order to synthesise oligosaccharides of biological interest or to develop vaccines against dental caries, elucidation of the factors determining the regiospecificity and the regioselectivity of glucansucrases is necessary. The cloning of glucansucrase encoding genes in addition to structure-function relationship studies have allowed the identification of important amino acid residues and have shown that glucansucrases are composed of two functional domains: a core region (ca. 1000 amino acids) involved in sucrose binding and splitting and a C-terminal domain (ca. 500 amino acids) composed of a series of tandem repeats involved in glucan binding. Enzymology studies have enabled different models for their action mechanism to be proposed. The use of secondary structure prediction has led to a clearer knowledge of structure-function relationships of glucansucrases. However, mainly due to the large size of these enzymes, data on the three-dimensional structure of glucansucrases (given by crystallography and modelling) remain necessary to clearly identify those features which determine function.
Asunto(s)
Glicósido Hidrolasas/fisiología , Glicosiltransferasas , Secuencia de Aminoácidos , Cariogénicos/metabolismo , Genes Bacterianos , Glucanos/metabolismo , Glucosiltransferasas/metabolismo , Glicósido Hidrolasas/química , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismo , Leuconostoc/enzimología , Datos de Secuencia Molecular , Oligosacáridos/metabolismo , Alineación de Secuencia , Streptococcus/enzimología , Streptococcus/genética , Relación Estructura-ActividadRESUMEN
An alpha-hydroxy acid derivative, alpha-butylglucoside lactate, was successfully prepared by enzymatic transesterification of alpha-butylglucoside with a lactate alkyl ester in a non-aqueous medium using immobilized lipase as biocatalyst. Ester synthesis in organic solvent was optimized. Solvent choice was made on the basis of substrate solubility and enzyme stability in the medium. A solvent-free reaction using butyllactate as lactate donor led to the highest yields. In the presence of 0.5M alphabutylglucoside and 100 g/L Novozym(R), a 67 % yield could be obtained within 40 h at 50 degrees C. However, the presence of butanol by-product limited the reaction to a maximum that could not be exceeded in closed systems. The elimination of the alcohol under reduced pressure resulted in the complete equilibrium shift of the transesterification reaction in favor of synthesis; below 15 mbars, more than 95% of 0.5M alpha-butylglucoside could be converted within 30 h. Moreover, simultaneous evaporation of water allowed hydrolysis of butyllactate to be eliminated. Consequently, a very high alpha-butylglucoside lactate concentration (170 g/) could be obtained in a single batch reaction. A single purification procedure, consisting of butyllactate extraction with hexane, enabled the product to be obtained at a purity above 95% (w/w). 1H and 13C NMR analysis later demonstrated that lactic acid was exclusively grafted onto the primary hydroxyl group of alphabutylglucoside.
Asunto(s)
Glucósidos/metabolismo , Lactatos/metabolismo , Azúcares Ácidos/metabolismo , Reactores Biológicos , Candida/enzimología , Enzimas Inmovilizadas , Lipasa/aislamiento & purificación , Lipasa/metabolismo , Espectroscopía de Resonancia Magnética , Presión , Rhizomucor/enzimología , Solventes , Azúcares Ácidos/química , Temperatura , AguaRESUMEN
The Neisseria polysaccharea gene encoding amylosucrase was subcloned and expressed in Escherichia coli. Sequencing revealed that the deduced amino acid sequence differs significantly from that previously published. Comparison of the sequence with that of enzymes of the alpha-amylase family predicted a (beta/alpha)8-barrel domain. Six of the eight highly conserved regions in amylolytic enzymes are present in amylosucrase. Among them, four constitute the active site in alpha-amylases. These sites were also conserved in the sequence of glucosyltransferases and dextransucrases. Nevertheless, the evolutionary tree does not show strong homology between them. The amylosucrase was purified by affinity chromatography between fusion protein glutathione S-transferase-amylosucrase and glutathione-Sepharose 4B. The pure enzyme linearly elongated some branched chains of glycogen, to an average degree of polymerization of 75.
Asunto(s)
Glucosiltransferasas/química , Glucosiltransferasas/genética , Neisseria/enzimología , Neisseria/genética , Estructura Secundaria de Proteína , Secuencia de Aminoácidos , Secuencia de Bases , Cromatografía de Afinidad , Clonación Molecular , Secuencia Conservada , Cartilla de ADN , Escherichia coli , Evolución Molecular , Glucosiltransferasas/metabolismo , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Alineación de SecuenciaRESUMEN
Dextransucrase (DSR-S) from Leuconostoc mesenteroides NRRL B-512F is a glucosyltransferase that catalyzes synthesis of soluble dextran from sucrose. In the presence of efficient acceptor molecules, such as maltose, the reaction pathway is shifted toward glucooligosaccharide synthesis. Like glucosyltransferases from oral streptococci, DSR-S possesses a C-terminal glucan-binding domain composed of a series of tandem repeats. In order to determine the role of the C-terminal region of DSR-S in dextran or oligosaccharide synthesis, four DSR-S genes with deletions at the 3' end were constructed. The results showed that the C-terminal region modulated the initial velocity of dextran synthesis but that the K(m) for sucrose, the optimum pH, and the activation energy were all unaffected by the deletions. The C-terminal domain modulated the rate of oligosaccharide synthesis whatever acceptor molecule was used (a good acceptor molecule such as maltose or a poor acceptor molecule such as fructose). The C-terminal domain seemed to play no role in the catalytic process in dextran and oligosaccharide synthesis. In fact, it seems that the role of the C-terminal domain of DSR-S may be to facilitate the translation of dextran and oligosaccharides from the catalytic site.
Asunto(s)
Dextranos/biosíntesis , Glucosiltransferasas/química , Leuconostoc/enzimología , Oligosacáridos/biosíntesis , Secuencia de Aminoácidos , Disacáridos/biosíntesis , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Maltosa/farmacología , Datos de Secuencia Molecular , Relación Estructura-Actividad , TemperaturaRESUMEN
The coding region for a novel Leuconostoc mesenteroides NRRL B-1299 dextransucrase gene (dsrB) was isolated and sequenced. Using degenerate primers homologous to a conserved region present in dextransucrases from Streptococcus (GTFs) and L. mesenteroides NRRL B-512F (DSRS) and conserved amino acid sequences located in the N-terminal catalytic region of these enzymes, about 60% of the DSRB encoding gene was isolated. Two sites, BamHI and HindIII, were identified which allowed one 0.5-kbp probe to be obtained to isolate the 5' and the 3' ends of dsrB. The nucleotide sequence of the dsrB gene was determined and found to consist of an open reading frame (ORF) of 4521 base pairs (bp) coding for a 1507-amino acid protein with an M1 of 168,511. The amino acid sequence is very close to that of DSRS. Like DSRS, the dextran produced appeared to be composed of only alpha (1-6) glucosidic bonds, and the oligosaccharides synthesized in the presence of acceptor maltose were also composed of alpha (1-6) linked glucosyl residues in addition to the maltosyl residue. DSRB thus appears to be a novel dextransucrase from L. mesenteroides NRRL B-1299. DSRB produces a dextran different from the typical dextran containing alpha (1-6) and alpha (1-2) linkages produced when this strain is grown in the presence of sucrose.
Asunto(s)
Genes Bacterianos , Glucanos/biosíntesis , Glucosiltransferasas/genética , Leuconostoc/genética , Secuencia de Aminoácidos , Clonación Molecular , Escherichia coli/genética , Leuconostoc/enzimología , Datos de Secuencia MolecularRESUMEN
When grown in glucose or fructose medium in the absence of sucrose, Leuconostoc mesenteroides NRRL B-1299 produces two distinct extracellular dextransucrases named glucose glucosyltransferase (GGT) and fructose glucosyltransferase (FGT). The production level of GGT and FGT is 10 to 20 times lower than that of the extracellular dextransucrase sucrose glucosyltransferase (SGT) produced on sucrose medium (traditional culture conditions). GGT and FGT were concentrated by ultrafiltration before sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Their molecular masses were 183 and 186 kDa, respectively, differing from the 195 kDa of SGT. The structural analysis of the dextran produced from sucrose and of the oligosaccharides synthesized by acceptor reaction in the presence of maltose showed that GGT and FGT are two different enzymes not previously described for this strain. The polymer synthesized by GGT contains 30% alpha(1-->2) linkages, while FGT catalyzes the synthesis of a linear dextran only composed of alpha(1-->6) linkages.
RESUMEN
Dextransucrase (DSRS) from Leuconostoc mesenteroides NRRL B-512F is a glucosyltransferase that catalyzes the synthesis of soluble dextran from sucrose or oligosaccharides when acceptor molecules, like maltose, are present. The L. mesenteroides NRRL B-512F dextransucrase-encoding gene (dsrS) was amplified by the polymerase chain reaction and cloned in an overexpression plasmid. The characteristics of DSRS were found to be similar to the characteristics of the extracellular dextransucrase produced by L. mesenteroides NRRL B-512F. The enzyme also exhibited a high homology with other glucosyltransferases. In order to identify critical amino acid residues, the DSRS sequence was aligned with glucosyltransferase sequences and four amino acid residues were selected for site-directed mutagenesis experiments: aspartic acid 511, aspartic acid 513, aspartic acid 551 and histidine 661. Asp-511, Asp-513 and Asp-551 were independently replaced with asparagine and His-661 with arginine. Mutation at Asp-511 and Asp-551 completely suppressed dextran and oligosaccharide synthesis activities, showing that at least two carboxyl groups (Asp-511 and Asp-551) are essential for the catalysis process. However, glucan-binding properties were retained, showing that DSRS has a two-domain structure like other glucosyltransferases. Mutations at Asp-513 and His-661 resulted in greatly reduced dextransucrase activity. According to amino acid sequence alignments of glucosyltransferases, alpha-amylases or cyclodextrin glucanotransferases, His-661 may have a hydrogen-bonding function.
Asunto(s)
Proteínas Bacterianas/aislamiento & purificación , Glucosiltransferasas/aislamiento & purificación , Leuconostoc/enzimología , Secuencia de Aminoácidos , Ácido Aspártico/química , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Catálisis , Dextranos/metabolismo , Escherichia coli , Genes Bacterianos , Glucosiltransferasas/química , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Histidina/química , Enlace de Hidrógeno , Leuconostoc/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , Sacarosa/metabolismoRESUMEN
The glucooligosaccharides (GOS), produced by Leuconostoc mesenteroides NRRL B-1299 dextransucrase through an acceptor reaction with maltose and sucrose, were purified by reverse phase chromatography. Logarithmic plots of retention time vs. dp of the GOS gave three parallel lines suggesting the existence of at least three families of homologous molecules. The structure (13C and 1H NMR spectroscopy) and reactivity of the purified molecules of the three families were investigated. All the products bear a maltose residue at the reducing end. The GOS in the first family (named OD) contained additional glucosyl residues all alpha-(1-->6) linked. The smallest molecule in this first series was panose or alpha-D-glucopyranosyl-(1-->6)-D-maltose (dp 3). All the OD molecules were shown to be good acceptors for dextransucrase in the presence of sucrose. The second family, named R, was composed of linear GOS containing alpha-(1-->6)-linked glucosyl residues and a terminal alpha-(1-->2)-linked residue at the non-reducing end of the molecule; the smallest molecule in this family was alpha-D-glucopyranosyl-(1-->2)-D-panose (dp 4). The third family, R', was formed of GOS containing additional residues linked through alpha-(1-->6) linkages that constitute the linear chain, and an alpha-(1-->2)-branched residue located on the penultimate element of the chain, near the non-reducing end. The smallest molecule in this series is alpha-D-glucopyranosyl-(1-->6)-[alpha-D-glucopyranosyl-(1-->2)]-alpha-D- glucopyranosyl-(1-->6)-D-panose, dp 6. R and R' GOS are very poor acceptors for L. mesenteroides NRRL B-1299 dextransucrase. This study makes it possible to suggest a rather simple reaction scheme, where molecules Ri, R'i and ODi of the same dp all result from the glucosylation of the same GOS: ODi-l.
Asunto(s)
Glucosiltransferasas/metabolismo , Leuconostoc/enzimología , Oligosacáridos/síntesis química , Secuencia de Carbohidratos , Glucosa/metabolismo , Glucósidos/síntesis química , Espectroscopía de Resonancia Magnética , Maltosa/metabolismo , Datos de Secuencia Molecular , Sacarosa/metabolismoRESUMEN
The coding region for a Leuconostoc mesenteroides NRRL B-1299 dextransucrase gene (dsrA) was isolated and sequenced. Using a pair of primers designed on the basis of two highly conserved amino-acid (aa) sequences in L. mesenteroides NRRL B-512F dextransucrase and streptococcal glucosyltransferases (GTFs), a fragment of dsrA was amplified by the polymerase chain reaction (PCR). This PCR product was used as an hybridization probe to isolate a 1.8-kb fragment identified as the central region of dsrA. Cleavage by Sac I of this fragment allowed two probes to be obtained to isolate the 5' and the 3' ends of dsrA. The nucleotide sequence of the dsrA gene was determined and found to consist of an open reading frame (ORF) of 4870 base pairs (bp) coding for a 1290-aa protein with an M(r) of 145590. The aa sequence exhibited a high similarity with other GTFs. The two domains previously described in GTFs are conserved in DSRA: an N-terminal conserved domain and a C-terminal domain composed of a series of repeats. Surprisingly, the expected signal peptide was not detected. The entire gene was reconstructed and the activity of DSRA was investigated. The dextran produced appeared to be composed of 85% alpha (1-6) and 15% alpha (1-3) linkages and the oligosaccharides synthesized in the presence of maltose were mainly composed of alpha (1-6) linkages. This enzyme is a novel dextransucrase from L. mesenteroides NRRL B-1299 producing no alpha (1-2) linkages and is the first glucosyltransferase having no signal peptide described.