246 research outputs found

    Development of a multilocus sequence typing method for analysis of Lactobacillus plantarum strains

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    Lactobacillus plantarum is a species of considerable industrial and medical interest. To date, the lack of reliable molecular methods for definite identification at strain level has hindered studies of the population biology of this organism. Here, a multilocus sequence typing (MLST) system for this organism is described, which exploits the genetic variation present in six housekeeping loci to determine the genetic relationship among isolates. The MLST system was established using 16 L. plantarum strains that were also characterized by ribotyping and restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified 16S–23S rDNA intergenic spacer region (ISR). Ribotyping grouped the strains into four groups; however, RFLP analysis of the ISRs showed no differences in the strains analysed. In contrast, MLST had a good discriminatory ability. The sequence analysis of the six genes showed 14 different allelic combinations, with 12 of them represented by only one strain. By using this MLST approach we were able to confirm the identity of two strains deposited in the Spanish Type Culture Collection as different strains. Phylogenetic analysis indicated a panmictic population structure of L. plantarum and split decomposition analysis indicated that recombination plays a role in creating genetic heterogeneity in L. plantarum. As MLST allows precise identification, and easy comparison and exchange of results obtained in different laboratories, the future application of this new molecular method could be useful for the identification of valuable L. plantarum strainsThis work has been supported by grants AGL2005-00470 and RM03- 002. We thank R. Gonza´lez and E. Garcı´a for their critical reading of the manuscript. The technical assistance of M. V. Santamarı´a is greatly appreciated. We also thank A. Hexter for correcting the English version. B. de las Rivas is a recipient of a postdoctoral fellowship and A. Marcobal of a predoctoral fellowship both from the Comunidad de Madrid.Peer reviewe

    Degradación de compuestos fenólicos presentes en vino mediante cepas de Lactobacillus plantarum

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    Trabajo presentado al IX Congreso Nacional de Investigación Enológica (GIENOL) celebrado en Badajoz del 29 de mayo al 1 de junio de 2007.Los compuestos fenólicos influyen en las características sensoriales y nutricionales del vino. La especie Lactobacillus plantarum, modelo de cultivo iniciador en biotecnología de alimentos vegetales, es una de las especies de bacterias lácticas mayoritarias en vino. El estudio pretende analizar la capacidad que presentan varias cepas de L. plantarum para degradar 15 de los compuestos fenólicos mayoritarios presentes en vinos.Este trabajo ha sido realizado gracias a la financiación recibida de los proyectos AGL2005-00470 (CICYT), RM03-002 (INIA) y 07G/0035/2003 (CAM)Peer reviewe

    Characterization of a benzyl alcohol dehydrogenase from Lactobacillus plantarum WCFS1

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    Aroma is an important sensory parameter of food products. Lactic acid bacteria have enzymatic activities that could be important in the modification of food aroma. The complete genome sequence from Lactobacillus plantarum WCFS1 shows a gene (lp_3054) putatively encoding a protein with benzyl alcohol dehydrogenase activity. To confirm its enzymatic activity lp_3054 from this strain has been overexpressed and purified. Protein alignment indicated that lp_3054 is a member of the family of NAD(P)-dependent long-chain zinc-dependent alcohol dehydrogenases. In lp_3054 all of the residues involved in zinc and cofactor binding are conserved. It is also conserved the residue that determines the specificity of the dehydrogenase toward NAD+ rather than NADP+ and, therefore, L. plantarum benzyl alcohol dehydrogenase is less active in the presence of NADP+ than in the presence of NAD+. The purified enzyme exhibits optimal activity at pH 5.0 and 30 °C. The kinetic parameters Km and Vmax on benzyl alcohol as a substrate were, respectively, 0.23 mM and 204 μmol h-1 mg-1. Besides its activity toward benzyl alcohol, it showed activity against nerol, geraniol, phenethyl alcohol, cinnamyl alcohol, and coniferyl alcohol, all of which are volatile compounds involved in determining food aroma. The biochemical demonstration of a functional benzyl alcohol dehydrogenase activity in this lactic acid bacteria species should be considered when the influence of bacterial metabolism in the aroma of food products is determinedL. plantarum WCFS1 strain was kindly provided by M. Kleerebezem from the Wageningen Centre for Food Sciences, NIZO Food Research. The technical assistance of M. V. Santamaría is greatly appreciatedPeer reviewe

    The pURI family of expression vectors: A versatile set of ligation independent cloning plasmids for producing recombinant His-fusion proteins

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    A family of restriction enzyme- and ligation-independent cloning vectors has been developed for producing recombinant His-tagged fusion proteins in Escherichia coli. These are based on pURI2 and pURI3 expression vectors which have been previously used for the successful production of recombinant proteins at the milligram scale. The newly designed vectors combines two different promoters (lppp-5 and T7 RNA polymerase Ø10), two different endoprotease recognition sites for the His6-tag removal (enterokinase and tobacco etch virus), different antibiotic selectable markers (ampicillin and erythromycin resistance), and different placements of the His 6-tag (N- and C-terminus). A single gene can be cloned and further expressed in the eight pURI vectors by using six nucleotide primers, avoiding the restriction enzyme and ligation steps. A unique NotI site was introduced to facilitate the selection of the recombinant plasmid. As a case study, the new vectors have been used to clone the gene coding for the phenolic acid decarboxylase from Lactobacillus plantarum. Interestingly, the obtained results revealed markedly different production levels of the target protein, emphasizing the relevance of the cloning strategy on soluble protein production yield. Efficient purification and tag removal steps showed that the affinity tag and the protease cleavage sites functioned properly. The novel family of pURI vectors designed for parallel cloning is a useful and versatile tool for the production and purification of a protein of interest. © 2010 Elsevier Inc. All rights reserved.This work was supported by grants RM2008-00002 (Instituto Nacional de Investigación Agraria y Alimentaría), AGL2008-01052, Consolider INGENIO 2010 CSD2007-00063 FUN-C-FOOD (Comisión Interministerial de Ciencia y Tecnología), and S-0505/AGR/000153 and S2009/AGR-1469 (ALIBIRD) (Comunidad de Madrid). J.M.M. thanks the Ministerio de Ciencia e Innovación for a research grant (BFU2007-67404/BMC) and “Factoría de Cristalización” Consolider-Ingenio 2010 in support of his research. The technical assistance of M.V. Santamaría is greatly appreciated. J.A. Curiel is a recipient of a predoctoral fellowship from the MEC.Peer Reviewe

    Study of the inhibitory activity of phenolic compounds found in olive products and their degradation by Lactobacillus plantarum strains

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    Lactobacillus plantarum is the main species responsible for the spontaneous fermentation of Spanish-style green olives. Olives and virgin oil provide a rich source of phenolic compounds. This study was designed to evaluate inhibitory growth activities of nine olive phenolic compounds against four L. plantarum strains isolated from different sources, and to explore the L. plantarum metabolic activities against these phenolic compounds. None of the nine compounds assayed (oleuropein, hydroxytyrosol, tyrosol, as well as vanillic, p-hydroxybenzoic, sinapic, syringic, protocatechuic and cinnamic acids) inhibited L. plantarum growth at the concentration found in olive products. Oleuropein and tyrosol concentrations higher than 100 mM were needed to inhibit L. plantarum growth. On the other hand, sinapic and syringic acid showed the highest inhibitory activity since concentrations ranging from 12.5 to 50 mM inhibited L. plantarum growth in all the strains analyzed. Among the nine compounds assayed, only oleuropein and protocatechuic acid were metabolized by L. plantarum strains grown in the presence of these compounds. Oleuropein was metabolized mainly to hydroxytyrosol, while protocatechuic acid was decarboxylated to catechol. Metabolism of oleuropein was carried out by inducible enzymes since a cell-free extract from a culture grown in the absence of oleuropein was unable to metabolize it. Independent of their isolation source, the four L. plantarum strains analysed showed similar behaviour in relation to the inhibitory activity of phenolic compounds, as well as their ability to metabolize these compoundsThis work was supported by grants AGL2005-00470 (CICYT), FUN-C-FOOD Consolider 25506 (MEC), RM03-002 (INIA), and S-0505/AGR/000153 (CAM). J.M. Landete and J. A. Curiel are recipients of a postdoctoral and predoctoral fellowship, respectively, from the MEC. H. Rodríguez is a recipient of a predoctoral fellowship from the I3P-CSIC.Peer reviewe

    The Lp_3561 and Lp_3562 enzymes support a functional divergence process in the lipase/esterase toolkit from Lactobacillus plantarum

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    Lactobacillus plantarum species is a good source of esterases since both lipolytic and esterase activities have been described for strains of this species. No fundamental biochemical difference exists among esterases and lipases since both share a common catalytic mechanism. L. plantarum WCFS1 possesses a protein, Lp_3561, which is 44% identical to a previously described lipase, Lp_3562. In contrast to Lp_3562, Lp_3561 was unable to degrade esters possessing a chain length higher than C4 and the triglyceride tributyrin. As in other L. plantarum esterases, the electrostatic potential surface around the active site in Lp_3561 is predicted to be basic, whereas it is essentially neutral in the Lp_3562 lipase. The fact that the genes encoding both proteins were located contiguously in the L. plantarum WCFS1 genome, suggests that they originated by tandem duplication, and therefore are paralogs as new functions have arisen during evolution. The presence of the contiguous lp_3561 and lp_3562 genes was studied among L. plantarum strains. They are located in a 8,903 bp DNA fragment that encodes proteins involved in the catabolism of sialic acid and are predicted to increase bacterial adaptability under certain growth conditions

    The crystal structure of galactitol-1-phosphate 5-dehydrogenase from Escherichia coli K12 provides insights into its anomalous behavior on IMAC processes

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    AbstractEndogenous galactitol-1-phosphate 5-dehydrogenase (GPDH) (EC 1.1.1.251) from Escherichia coli spontaneously interacts with Ni2+-NTA matrices becoming a potential contaminant for recombinant, target His-tagged proteins. Purified recombinant, untagged GPDH (rGPDH) converted galactitol into tagatose, and d-tagatose-6-phosphate into galactitol-1-phosphate, in a Zn2+- and NAD(H)-dependent manner and readily crystallized what has permitted to solve its crystal structure. In contrast, N-terminally His-tagged GPDH was marginally stable and readily aggregated. The structure of rGPDH revealed metal-binding sites characteristic from the medium-chain dehydrogenase/reductase protein superfamily which may explain its ability to interact with immobilized metals. The structure also provides clues on the harmful effects of the N-terminal His-tag.Structured summary of protein interactionsGPDH and GPDH bind by molecular sieving (View interaction)GPDH and GPDH bind by x-ray crystallography(View interaction)GPDH and GPDH bind by cosedimentation in solution (View interaction

    A diverse range of human gut bacteria have the potential to metabolize the dietary component gallic acid

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    The human gut microbiota contains a broad variety of bacteria that possess functional genes, with resultant metabolites that affect human physiology and therefore health. Dietary gallates are phenolic components that are present in many foods and beverages and are regarded as having health-promoting attributes. However, the potential for metabolism of these phenolic compounds by the human microbiota remains largely unknown. The emergence of high-throughput sequencing (HTS) technologies allows this issue to be addressed. In this study, HTS was used to assess the incidence of gallate-decarboxylating bacteria within the gut microbiota of healthy individuals for whom bacterial diversity was previously determined to be high. This process was facilitated by the design and application of degenerate PCR primers to amplify a region encoding the catalytic C subunit of gallate decarboxylase (LpdC) from total metagenomic DNA extracted from human fecal samples. HTS resulted in the generation of a total of 3,261,967 sequence reads and revealed that the primary gallate-decarboxylating microbial phyla in the intestinal microbiota were Firmicutes (74.6%), Proteobacteria (17.6%), and Actinobacteria (7.8%). These reads corresponded to 53 genera, i.e., 47% of the bacterial genera detected previously in these samples. Among these genera, Anaerostipes and Klebsiella accounted for the majority of reads (40%). The usefulness of the HTS-lpdC method was demonstrated by the production of pyrogallol from gallic acid, as expected for functional gallate decarboxylases, among representative strains belonging to species identified in the human gut microbiota by this method. Importance: Despite the increasing wealth of sequencing data, the health contributions of many bacteria found in the human gut microbiota have yet to be elucidated. This study applies a novel experimental approach to predict the ability of gut microbes to carry out a specific metabolic activity, i.e., gallate metabolism. The study showed that, while gallate-decarboxylating bacteria represented 47% of the bacterial genera detected previously in the same human fecal samples, no gallate decarboxylase homologs were identified from representatives of Bacteroidetes. The presence of functional gallate decarboxylases was demonstrated in representative Proteobacteria and Firmicutes strains from the human microbiota, an observation that could be of considerable relevance to the in vivo production of pyrogallol, a physiologically important bioactive compound

    Identificación y caracterización de esterasas de Lactobacillus plantarum con interés en tecnología de alimentos

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    Resumen del trabajo presentado a la 7ª Reunión de la Red Tematica Bal: "Participación de las Bacterias Lácticas en la Salud Humana y en la Calidad Alimentaria" celebrado en Madrid del 4 al 5 de julio de 2013.Los ésteres son compuestos aromáticos que a pesar de encontrarse en niveles traza, son muy importantes en el perfil aromático de los alimentos. Pequeñas variaciones en los niveles de estos compuestos pueden tener importantes efectos en el aroma, y por lo tanto en la calidad, de los alimentos. Los ésteres de los alimentos se originan por acción de enzimas con actividad esterasa (EC 3.1.1.x) que pueden catalizar tanto reacciones de hidrólisis como de síntesis en función de las condiciones de reacción, por lo que tienen un gran interés en biotecnología. Entre estas enzimas se pueden distinguir carboxilesterasas, arilesterasas y lipasas. Las carboxilesterasas y las arilesterasas catalizan la hidrólisis de ésteres de cadena alifática corta o media solubles en agua, mientras que las lipasas presentan actividad frente a ésteres de cadena larga e insolubles en agua. Las feruloil esterasas, son un tipo de arilesterasas capaces de hidrolizar el enlace éster entre los ácidos cinámicos y los azúcares de las paredes celulares vegetales liberando compuestos fenólicos como el ácido cafeico, p-cumárico y ferúlico, los cuales presentan numerosas aplicaciones en la industria alimentaría. Lactobacillus plantarum es la especie de bacteria láctica modelo en fermentaciones de sustratos vegetales, en donde los ésteres de compuestos fenólicos, se encuentran en altas concentraciones. En el genoma de L. plantarum aparecen anotados genes que codifican “esterasas” o “lipasas” cuya funcionalidad no se ha comprobado bioquímicamente a pesar del interés biotecnológico que presentan. Por ello el objetivo de este trabajo es la identificación y caracterización de posibles esterasas en L. plantarum WCFS1. A pesar de que se han clonado los genes que codifican 14 posibles esterasas o lipasas, sólo se han podido producir y purificar correctamente las proteínas Lp_0796, Lp_0973, Lp_1002, Lp_2923, Lp_2987, Lp_3561 y Lp_3562. Utilizando ésteres derivados de p-nitrofenilo que varían en la longitud de su cadena alifática (desde acetato de p-nitrofenilo hasta palmitato de pnitrofenilo) se ha comprobado que todas las proteínas estudiadas hidrolizan mejor los ésteres de cadena corta, aunque Lp_1002, Lp_2926 y Lp_3562 también hidrolizan eficazmente palmitato de p-nitrofenilo.La proteína Lp_2987 es la única que no presenta actividad sobre ninguno de los derivados ensayados por lo que no se puede considerar como “esterasa”. La especificidad de substrato de las esterasas también se ha evaluado mediante una colección de ésteres que permite conocer su selectividad respecto a la carga del substrato, al tamaño de la cadena o al alcohol presente. Todas las proteínas purificadas, excepto Lp_2987, presentan actividad sobre acetato de fenilo, por lo que se pueden considerar como “aril esterasas”. La proteína Lp_0973 es la única que, además de acetato de fenilo, degrada triacetina y tributirina. De las esterasas estudiadas, la proteína Lp_0796 es la más interesante puesto que es una “feruloil esterasa” que hidroliza ésteres de ácidos hidroxicinámicos, siendo la primera vez que se describe una proteína con esta actividad en L. plantarum. Con objeto de mejorar la actividad de las esterasas para su posible uso industrial se han realizado experimentos de cristalización, inmovilización y evolución dirigida de alguna de ellas. Los resultados obtenidos indican que L. plantarum es una fuente adecuada de enzimas con actividad esterasa de gran influencia en el aroma de los alimentos.Peer reviewe

    Food phenolics and Lactiplantibacillus plantarum

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    17 Pág.Phenolic compounds are important constituents of plant food products. These compounds play a key role in food characteristics such as flavor, astringency and color. Lactic acid bacteria are naturally found in raw vegetables, being Lactiplantibacillus plantarum the most commonly used commercial starter for the fermentation of plant foods. Hence, the metabolism of phenolic compounds of L. plantarum has been a subject of study in recent decades. Such studies confirm that L. plantarum, in addition to presenting catalytic capacity to transform aromatic alcohols and phenolic glycosides, exhibits two main differentiated metabolic routes that allow the biotransformation of dietary hydroxybenzoic and hydroxycinnamic acid-derived compounds. These metabolic pathways lead to the production of new compounds with new biological and organoleptic properties. The described metabolic pathways involve the action of specialized esterases, decarboxylases and reductases that have been identified through genetic analysis and biochemically characterized. The purpose of this review is to provide a comprehensive and up-to-date summary of the current knowledge of the metabolism of food phenolics in L. plantarum.This work was financially supported by grants AGL2005-00470, AGL2008-01052, AGL2011-22745, AGL2014-52911-R, and AGL2017-84614-C2-2-R funded by MCIN/AEI/10.13039/501100011033, CSIC-202240I177 and by ERDF A way of making Europe. Ana Sánchez Arroyo is a recipient of the PRE2018-083862 FPI contract funded by MCIN/AEI/10.13039/501100011033 and by ESF Investing in your future.Peer reviewe
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