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The synthesis of β-galactosyl xylitol derivatives using immobilized LacA β-galactosidase from

This work addresses the amino acid sequence, structural analysis, biochemical characterization and glycosidase activity of two recombinant α-rhamnosidases, Ram1 and Ram2, from Lactobacillus plantarum WCFS1. The substrate specificity of both enzymes towards the disaccharide rutinose and natural dietary flavonoids naringin and rutin was also determined and compared to that of a commercial multienzyme complex (Pectinex Ultra Passover, PPO). Ram1 is a less acidic- and heat-active enzyme than Ram2 and exhibited a high activity towards pNP-α-L-rhamnopyranoside, but it was unable to hydrolyze neither rutinose, naringin or rutin. In contrast, Ram2 enzyme showed a substrate specificity towards α-(1➔6) glycosidic flavonoids, such as rutin, and the disaccharide rutinose. The mechanism of action of Ram2 towards rutin was elucidated and revealed the potential cost-effective and selective production of the monoglycosylated flavonoid isoquercetin (quercetin-3-O-glucoside). PPO efficiently converted both naringin and rutin into their corresponding aglycones. These findings revealed the potential usefulness of PPO for the improvement of sensory properties of beverages through debittering of citrus juices, as well as the potential use of Ram2 to selectively produce isoquercetin, a highly valued and bioactive flavonoid whose production is not currently affordable., This work was supported by the Spanish Ministry of Economy, Industry and Competitiveness (Projects AGL2017-84614-C2-1-R and AGL2017-84614-C2-2-R).

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This work describes the high capacity of MelA α-galactosidase from

In Lactobacillus plantarum the metabolism of hydroxybenzoic and hydroxycinnamic acid derivatives follows a similar two-step pathway, an esterase action followed by a decarboxylation. The L. plantarum esterase genes involved in these reactions have been cloned into pNZ8048 or pT1NX plasmids and transformed into technologically relevant lactic acid bacteria. None of the strains assayed can hydrolyse methyl gallate, a hydroxybenzoic ester. The presence of the L. plantarum tannase encoding genes (tanALp or tanBLp) on these bacteria conferred their detectable esterase (tannase) activity. Similarly, on hydroxycinnamic compounds, esterase activity for the hydrolysis of ferulic acid was acquired by lactic acid bacteria when L. plantarum esterase (JDM1_1092) was present. This study showed that the heterologous expression of L. plantarum esterase genes involved in the metabolism of phenolic acids allowed the production of healthy compounds which increase the bioavailability of these dietary compounds in food relevant lactic acid bacteria., This work was supported by grants AGL2017-84614-C2-2-R and AGL2014-52911 (AEI/FEDER, UE) from the Agencia Estatal de Investigación (AEI) and RM2012-0004-00-00 and RTA2017-00002-00-00 from the Spanish Ministry of Science, Innovation and Universities.

Second Edition., Lactobacillus plantarum (currently Lactiplantibacillus plantarum) is the main bacterial species associated to olive processing. During the last years, numerous studies have been carried out on the metabolism of phenolics compounds by L. plantarum strains, due to the great importance of these compounds in the health beneficial properties of olive products. Among the olive phenolic acids studied, some hydroxycinnamic acids (caffeic, m-coumaric, o-coumaric, p-coumaric, ferulic, and sinapic acids) as well as some hydroxybenzoic acids (gallic and protocatechuic acids) were metabolized by L. plantarum. The decarboxylase and reductase enzymes responsible for these biochemical reactions have been identified, recombinantly produced, and biochemically characterized. Oleuropein, the main secoiridoid glucoside present in olives, is hydrolyzed by L. plantarum by the sequential action of unidentified enzymes, giving elenoic acid and hydroxytyrosol as end-products. It has been also studied the response of L. plantarum to some olive-related compounds (olive oil, p-coumaric acid, gallic acid, and oleuropein) by genome-scale transcriptomic signature and physiological analysis.

This work describes the high capacity of MelA α-galactosidase from Lactobacillus plantarum WCFS1 to transfer galactosyl residues from melibiose to the C6-hydroxyl group of disaccharide-acceptors with β-linkages (lactulose, lactose, and cellobiose) or α-linkages (isomaltulose and isomaltose) to produce novel galactose-containing hetero-oligosaccharides (HOS). A comprehensive nuclear magnetic resonance characterization of the transfer products derived from melibiose:lactulose reaction mixtures revealed the biosynthesis of α-d-galactopyranosyl-(1 → 6)-β-d-galactopyranosyl-(1 → 4)-β-d-fructose as the main component as well as the presence of α-d-galactopyranosyl-(1 → 3)-β-d-galactopyranosyl-(1 → 4)-β-d-fructose and α-d-galactopyranosyl-(1 → 6)-α-d-galactopyranosyl-(1 → 6)-β-d-galactopyranosyl-(1 → 4)-β-d-fructose. Melibiose-derived α-galactooligosaccharides (α-GOS), manninotriose and verbascotetraose, were also simultaneously synthesized. An in vitro assessment of the intestinal digestibility of the novel biosynthesized HOS revealed a high resistance of α-galactosides derived from lactulose, lactose, cellobiose, and isomaltulose. According to the evidence gathered for conventional α-GOS and certain disaccharides used as acceptors in this work, these novel nondigestible α-galactosides could be potential candidates to selectively modulate the gut microbiota composition, among other applications, such as low-calorie food ingredients., Paloma Delgado-Fernández thanks to Ministry of Science, Innovation, and Universities of Spain for providing the FPI predoctoral fellowship.

The production, biochemical characterization, and carbohydrate specificity of LacA β-galactosidase (locus

This comprehensive work addresses, for the first time, the heterologous production, purification, biochemical characterization and carbohydrate specificity of MelA, a cold-active α-galactosidase belonging to the Glycoside Hydrolase family 36, from the probiotic organism Lactobacillus plantarum WCFS1. The hydrolytic activity of MelA α-galactosidase on a wide range of p-nitrophenyl glycoside derivatives and carbohydrates of different molecular-weights showed its high selectivity and efficiency towards the α(1 → 6) glycosidic bonds involving the anomeric carbon of galactose and the C6-hydroxyl group of galactose or glucose units. MelA α-galactosidase also presented a high regioselectivity, efficiency and diversity in accommodating donor and acceptor substrates for the synthesis of α-GOS through transgalactosylation reactions. The catalytic mechanism of MelA for the production of α-GOS was elucidated, revealing its great preference for the transfer of galactosyl residues to the C6-hydroxyl group of galactose units to elongate the chain of α-GOS having either a terminal sucrose (raffinose family oligosaccharides, RFOS) or a terminal glucose (melibiose, manninotriose and verbascotetraose). Our findings indicate the feasibility of using MelA α-galactosidase from Lactobacillus plantarum WCFS1 in the hydrolysis of RFOS and in the efficient and versatile synthesis of α-GOS with appealing functional properties in the context of food and nutraceutical applications., This work has been financed by the Spanish Ministries of Economy, Industry and Competitiveness (Projects AGL2017-84614-C2-1-R and AGL2017-84614-C2-2-R) and Science, Innovation and Universities (Project RTI2018-101273-J-I00). Paloma Delgado-Fernández thanks to Ministry of Science, Innovation and Universities of Spain for providing FPI predoctoral fellowship.

[Background]: α-Amylases specifically catalyse the hydrolysis of the internal α-1, 4-glucosidic linkages of starch. Glycoside hydrolase (GH) family 13 is the main α-amylase family in the carbohydrate-active database. Lactobacillus plantarum WCFS1 possesses eleven proteins included in GH13 family. Among these, proteins annotated as maltose-forming α-amylase (Lp_0179) and maltogenic α-amylase (Lp_2757) were included., [Results]: In this study, Lp_0179 and Lp_2757 L. plantarum α-amylases were structurally and biochemically characterized. Lp_2757 displayed structural features typical of GH13_20 subfamily which were absent in Lp_0179. Genes encoding Lp_0179 (Amy2) and Lp_2757 were cloned and overexpressed in Escherichia coli BL21(DE3). Purified proteins showed high hydrolytic activity on pNP-α-D-maltopyranoside, being the catalytic efficiency of Lp_0179 remarkably higher. In relation to the hydrolysis of starch-related carbohydrates, Lp_0179 only hydrolysed maltopentaose and dextrin, demonstrating that is an exotype glucan hydrolase. However, Lp_2757 was also able to hydrolyze cyclodextrins and other non-cyclic oligo- and polysaccharides, revealing a great preference towards α-1,4-linkages typical of maltogenic amylases., [Conclusions]:The substrate range as well as the biochemical properties exhibited by Lp_2757 maltogenic α-amylase suggest that this enzyme could be a very promising enzyme for the hydrolysis of α-1,4 glycosidic linkages present in a broad number of starch-carbohydrates, as well as for the investigation of an hypothetical transglucosylation activity under appropriate reaction conditions., This work was financially supported by Grants AGL2017-84614-C2-1-R and AGL2017-84614-C2-2-R (AEI/FEDER, UE).

Oleuropein (OLE) is a secoiridoid unique to Oleaceae known to play a role in the plant–herbivore interaction. However, it is not clear how this molecule is induced to mediate plant responses to microbes and how microbes, in turn, withstand with OLE. To better understand how OLE affects the plant–microbe interaction, the contribution of differential gene expression in the adaptation to OLE was characterized by whole genome transcriptional profiling in Lactobacillus plantarum, a bacterium associated to the olive. OLE downregulated functions associated to rapid growth, remodeled membrane phospholipid biosynthesis pathways and markedly repressed the expression of several ABC transporters from L. plantarum. Genes encoding the plantaricin and lamABDCA quorum-sensing (QS) systems were down-regulated indicating the potential of OLE as a QS-antagonist. Notably, OLE diminished the expression of a set of genes encoding inmunomodulatory components and reoriented metabolic pathways to increase protein acetylation, probably to attenuate plant immunity. Responses were also triggered to repress the transport of acetoin and to buffer reactive oxygen species accumulation, two signals involved in plant development. The results suggest that OLE could act as a signaling molecule in the plant–microbe interaction and facilitate the accommodation of beneficial microbes such as L. plantarum by the plant host, via controlled expression of bacterial molecular players involved in this reciprocal interplay.

Oleuropein (OLE) is a secoiridoid unique to Oleaceae known to play a role in the plant¿herbivore interaction. However, it is not clear how this molecule is induced to mediate plant responses to microbes and how microbes, in turn, withstand with OLE. To better understand how OLE affects the plant¿microbe interaction, the contribution of differential gene expression in the adaptation to OLE was characterized by whole genome transcriptional profiling in Lactobacillus plantarum, a bacterium associated to the olive. OLE downregulated functions associated to rapid growth, remodeled membrane phospholipid biosynthesis pathways and markedly repressed the expression of several ABC transporters from L. plantarum. Genes encoding the plantaricin and lamABDCA quorum-sensing (QS) systems were down-regulated indicating the potential of OLE as a QS-antagonist. Notably, OLE diminished the expression of a set of genes encoding inmunomodulatory components and reoriented metabolic pathways to increase protein acetylation, probably to attenuate plant immunity. Responses were also triggered to repress the transport of acetoin and to buffer reactive oxygen species accumulation, two signals involved in plant development. The results suggest that OLE could act as a signaling molecule in the plant¿microbe interaction and facilitate the accommodation of beneficial microbes such as L. plantarum by the plant host, via controlled expression of bacterial molecular players involved in this reciprocal interplay., This work was supported by grants AGL2017-84614-C2-2-R and AGL2014-52911 (AEI/FEDER, UE).

Lactobacillus plantarum is the lactic acid bacterial species most frequently found in plant-food fermentations where hydroxycinnamic acids are abundant. L. plantarum efficiently decarboxylates these compounds and also reduces them, yielding substituted phenylpropionic acids. Although the reduction step is known to be induced by a hydroxycinnamic acid, the enzymatic machinery responsible for this reduction pathway has not been yet identified and characterized. A previous study on the transcriptomic response of L. plantarum to p-coumaric acid revealed a marked induction of two contiguous genes, lp_1424 and lp_1425, encoding putative reductases. In this work, the disruption of these genes abolished the hydroxycinnamate reductase activity of L. plantarum, supporting their involvement in such chemical activity. Functional in vitro studies revealed that Lp_1425 (HcrB) exhibits hydroxycinnamate reductase activity but was unstable in solution. In contrast, Lp_1424 (HcrA) was inactive but showed high stability. When the hcrAB genes were co-overexpressed, the formation of an active heterodimer (HcrAB) was observed. Since L. plantarum reductase activity was only observed on hydroxycinnamic acids (o-coumaric, m-coumaric, p-coumaric, caffeic, ferulic, and sinapic acids), the presence of a hydroxyl group substituent on the benzene ring appears to be required for activity. In addition, hydroxycinnamate reductase activity was not widely present among lactic acid bacteria, and it was associated with the presence of hcrAB genes. This study revealed that L. plantarum hydroxycinnamate reductase is a heterodimeric NADH-dependent coumarate reductase acting on a carbon-carbon double bond., This work was supported by grant AGL2014-52911-R (AEI/FEDER, UE) (MINEICO). L. Santamaría is a recipient of an FPI Fellowship from the MINEICO.

© 2014 Elsevier Ltd. Lactobacillus plantarum is a lactic acid bacteria found in fermented foods on which high salt concentrations are present. So far, no salt-tolerant lipase has been described from lactic acid bacteria. In the present study, the gene lp_3562 encoding a putative esterase/lipase was cloned and expressed in Escherichia coli BL21 (DE3) and the overproduced Lp_3562 protein has been biochemically characterized. Lp_3562 is a lipase active on tributyrin and also on other long chain substrates. Although the highest activity was observed at pH 7, the enzyme showed activity at pH 5.0-8.0. Lp_3562 exhibited optimal lipase activity at 40°C and, showing more than 40% of maximal activity at refrigeration temperature (5°C). Conversely, Lp_3562 presented good thermostability exhibiting more than 80% of its maximal activity after 20h incubation at 45°C. More interestingly, Lp_3562 was active under NaCl concentrations higher than 20%. The biochemical properties exhibited by Lp_3562 make this halotolerant lipase attractive for its use in food fermentations., This work was financially supported by grants AGL2011-22745 and BFU2010-17929 (MINECO), S2009/AGR-1469 (ALIBIRD) (Comunidad de Madrid), and RM2012-00004 (INIA).

[Scope]: This study was undertaken to expand our insights into the mechanisms involved in the tolerance to resveratrol (RSV) that operate at system-level in gut microorganisms and advance knowledge on new RSV-responsive gene circuits. [Methods and results]: Whole genome transcriptional profiling was used to characterize the molecular response of Lactobacillus plantarum WCFS1 to RSV. DNA repair mechanisms were induced by RSV and responses were triggered to decrease the load of copper, a metal required for RSV-mediated DNA cleavage, and HS, a genotoxic gas. To counter the effects of RSV, L. plantarum strongly up- or downregulated efflux systems and ABC transporters pointing to transport control of RSV across the membrane as a key mechanism for RSV tolerance. L. plantarum also downregulated tRNAs, induced chaperones, and reprogrammed its transcriptome to tightly control ammonia levels. RSV induced a probiotic effector gene and a likely deoxycholate transporter, two functions that improve the host health status. [Conclusion]: Our data identify novel protective mechanisms involved in RSV tolerance operating at system level in a gut microbe. These insights could influence the way RSV is used for a better management of gut microbial ecosystems to obtain associated health benefits., This work was supported by grant AGL2014-52911 (MINECO).

Lactobacillus plantarum is a lactic acid bacterium that can degrade food tannins by the successive action of tannase and gallate decarboxylase enzymes. In the L. plantarum genome, the gene encoding the catalytic subunit of gallate decarboxylase (lpdC, or lp_2945) is only 6.5 kb distant from the gene encoding inducible tannase (L. plantarum tanB [tanB], or lp_2956). This genomic context suggests concomitant activity and regulation of both enzymatic activities. Reverse transcription analysis revealed that subunits B (lpdB, or lp_0271) and D (lpdD, or lp_0272) of the gallate decarboxylase are cotranscribed, whereas subunit C (lpdC, or lp_2945) is cotranscribed with a gene encoding a transport protein (gacP, or lp_2943). In contrast, the tannase gene is transcribed as a monocistronic mRNA. Investigation of knockout mutations of genes located in this chromosomal region indicated that only mutants of the gallate decarboxylase (subunits B and C), tannase, GacP transport protein, and TanR transcriptional regulator (lp_2942) genes exhibited altered tannin metabolism. The expression profile of genes involved in tannin metabolism was also analyzed in these mutants in the presence of methyl gallate and gallic acid. It is noteworthy that inactivation of tanR suppresses the induction of all genes overexpressed in the presence of methyl gallate and gallic acid. This transcriptional regulator was also induced in the presence of other phenolic compounds, such as kaempferol and myricetin. This study complements the catalog of L. plantarum expression profiles responsive to phenolic compounds, which enable this bacterium to adapt to a plant food environment., N. Jiménez is a recipient of an FPI Fellowship from MINECO. This work was supported by grants AGL2014-52911-R, AGL2011-22745 (MINECO), and RM2008-00002 (Instituto Nacional de Investigación Agraria y Alimentaria).

Lactobacillus plantarum is frequently isolated from the fermentation of plant material where tannins are abundant. L. plantarum strains possess tannase activity to degrade plant tannins. An L. plantarum tannase (TanBLp, formerly called TanLp1) was previously identified and biochemically characterized. In this study, we report the identification and characterization of a novel tannase (TanALp). While all 29 L. plantarum strains analyzed in the study possess the tanBLp gene, the gene tanALp was present in only four strains. Upon methyl gallate exposure, the expression of tanBLp was induced, whereas tanALp expression was not affected. TanALp showed only 27% sequence identity to TanBLp, but the residues involved in tannase activity are conserved. Optimum activity for TanALp was observed at 30°C and pH 6 in the presence of Ca2+ ions. TanALp was able to hydrolyze gallate and protocatechuate esters with a short aliphatic alcohol substituent. Moreover, TanALp was able to fully hydrolyze complex gallotannins, such as tannic acid. The presence of the extracellular TanALp tannase in some L. plantarum strains provides them an advantage for the initial degradation of complex tannins present in plant environments. © 2014, American Society for Microbiology., This work was supported by grants AGL2011-22745, BFU2010-17929/BMC, Consolider INGENIO 2010 CSD2007-00063 FUN-C-FOOD (MINECO), S2009/AGR-1469 (ALIBIRD) (Comunidad de Madrid), and RM2012-00004 (Instituto Nacional de Investigación Agraria y Alimentaría). N. Jiménez is the recipient of an FPI fellowship from the MINECO.

Lactobacillus plantarum is frequently found in the fermentation of plant-derived food products, where hydroxycinnamoyl esters are abundant. L. plantarum WCFS1 cultures were unable to hydrolyze hydroxycinnamoyl esters; however, cell extracts from the strain partially hydrolyze methyl ferulate and methyl p-coumarate. In order to discover whether the protein Lp_0796 is the enzyme responsible for this hydrolytic activity, it was recombinantly overproduced and enzymatically characterized. Lp_0796 is an esterase that, among other substrates, is able to efficiently hydrolyze the four model substrates for feruloyl esterases (methyl ferulate, methyl caffeate, methyl p-coumarate, and methyl sinapinate). A screening test for the detection of the gene encoding feruloyl esterase Lp_0796 revealed that it is generally present among L. plantarum strains. The present study constitutes the description of feruloyl esterase activity in L. plantarum and provides new insights into the metabolism of hydroxycinnamic compounds in this bacterial species. © 2013, American Society for Microbiology., This work was supported by grants AGL2011-22745, BFU2010-17929/BMC, Consolider INGENIO 2010 CSD2007-00063 FUN-C-FOOD (MINECO), S2009/AGR-1469 (ALIBIRD) (Comunidad de Madrid), and RM2012-00004 (Instituto Nacional de Investigación Agraria y Alimentaría). We are grateful to M. V. Santamaría and J. M. Barcenilla. M. Esteban-Torres is a recipient of a JAE-predoctoral fellowship from the CSIC.

Póster presentado a la VII International Conference on Environmental Industrial and Applied Microbiology, celebrada en Madrid (España) del 18 al 20 de octubre de 2017., Ethylphenols represent a large family of substances, some of which possess a strong odorous activity which can influence the aroma of smoked food stuffs and numerous fermented beverages. These substances are naturally part of the composition of wines. Although grape musts contain only trace amounts of such phenolic compounds, red wines can have several hundred micrograms per liter. The precise origin of ethylphenols (4-ethylphenol and 4-ethylguaiacol) in red wines is not completely known. The presence of the yeast Brettanomyces bruxellensis has been correlated with an increase of ethylphenols in wine. However, initial works of Whiting and Carr showed that also certain bacteria (Lactobacillus pastorianus var. quinicus) are capable of decarboxylate and reduce cinnamic acids into their corresponding ethylphenols. More recent works, described that lactic acid bacteria belonging to the Lactobacillus plantarum species were also able to reduce the vinylphenols, produced by decarboxylation of hydroxycinnamic acids, to ethylphenols. In this work, the reductase involved in ethylphenol formation by L. plantarum WCFS1 was identified. Transcriptomic analysis in the presence of p-coumaric acid revealed in L. plantarum WCFS1 the induction of several genes annotated as putative reductases. The disruption of these genes allowed the identification of a gene which inactivation leads to the absence on vinilphenol reductase activity. The identified gene was cloned in E. coli. Contrarily to E. coli protein extracts, those extracts from E. coli overproducing the recombinant protein showed vinylphenol reductase activity. This result enables us to confirm that the produced protein was the protein involved on the reduction of vinylphenols in L. plantarum WCFS1., This work was supported by grant AGL2014-52911-R (MINEICO).

Statistics from structural genomics initiatives reveal that around 50–55% of the expressed, non-membrane proteins cannot be purified and therefore structurally characterized due to solubility problems, which emphasized protein solubility as one of the most serious concerns in structural biology projects. Lactobacillus plantarum CECT 748 produces an aggregation-prone glycosidase (LpBgl) that we crystallized previously. However, this result could not be reproduced due to protein instability and therefore further high-resolution structural analyses of LpBgl were impeded. The obtained crystals of LpBgl diffracted up to 2.48 Å resolution and permitted to solve the structure of the enzyme. Analysis of the active site revealed a pocket for phosphate-binding with an uncommon architecture, where a phosphate molecule is tightly bound suggesting the recognition of 6-phosphoryl sugars. In agreement with this observation, we showed that LpBgl exhibited 6-phospho-β-glucosidase activity. Combination of structural and mass spectrometry results revealed the formation of dimethyl arsenic adducts on the solvent exposed cysteine residues Cys211 and Cys292. Remarkably, the double mutant Cys211Ser/Cys292Ser resulted stable in solution at high concentrations indicating that the marginal solubility of LpBgl can be ascribed specifically to these two cysteine residues. The 2.30 Å crystal structure of this double mutant showed no disorder around the newly incorporated serine residues and also loop rearrangements within the phosphate-binding site. Notably, LpBgl could be prepared at high yield by proteolytic digestion of the fusion protein LSLt-LpBgl, which raises important questions about potential hysteretic processes upon its initial production as an enzyme fused to a solubility enhancer., We thank the ESRF (Grenoble, France) for provision of synchrotron radiation facilities (BM16 and ID23-2). Financial support from the Ministerio de Economía y Competitividad (BFU2010-17929/BMC) (to J.M.M.) and AGL2014-52911-R (to R.M.).

Dietary fats may exert selective pressures on Lactobacillus species, however, knowledge on the mechanisms of adaptation to fat stress in these organisms is still fragmentary. This study was undertaken to gain insight into the mechanisms of adaptation of Lactobacillus plantarum WCFS1 to olive oil challenge by whole genome transcriptional profiling using DNA microarrays. A set of 230 genes were differentially expressed by L. plantarum WCFS1 to respond to this vegetable oil. This response involved elements typical of the stringent response, as indicated by the induction of genes involved in stress-related pathways and downregulation of genes related to processes associated with rapid growth. A set of genes involved in the transport and metabolism of compatible solutes were downregulated, indicating that this organism does not require osmoprotective mechanisms in presence of olive oil. The fatty acid biosynthetic pathway was thoroughly downregulated at the transcriptional level, which coincided with a diminished expression of genes controlled by this pathway in other organisms and that are required for the respiratory function, pyruvate dehydrogenase activity, RNA processing and cell size setting. Finally, a set of genes involved in host-cell signaling by L. plantarum were differentially regulated indicating that olive oil can influence the expression of metabolic traits involved in the crosstalk between this bacterium and the host., This work was supported by grants AGL2014-52911 and AGL2011-22745 (MINECO).

Food phenolics exhibit a wide range of beneficial health effects due to their antioxidant properties. Natural phenolic compounds often occur as glycosides, esters, or polymers on which their bioactivity is reduced. Some food phenolics are transformed by the microbiota present during fermentation. This conversion is often essential for absorption and modulates the biological activity of these dietary compounds. Lactobacillus plantarum is the species most frequently used to ferment plant food products where phenolic compounds are abundant. The knowledge of the metabolism of phenolic compounds in L. plantarum is of interest as this bacterium possesses relevant enzymatic activities to obtain bioactive compounds. Complex phenolics are hydrolyzed by L. plantarum to simpler and more biologically active compounds. The complete enzymatic pathways for the biodegradation of polymeric hydroxybenzoic- and hydoxycinnamic-derived compounds are described. The adaptative behavior of L. plantarum under stress induced by phenolics also modulates traits beneficial for its gastrointestinal survival.

This article reports for the first time on the capacity of a wine Lactobacillus plantarum strain to alter the oxidative coloration of (þ)-catechin and (-)-epicatechin hydroethanolic wine model solutions in the presence of Fe2þ as catalyst. The time course of color development and pigment formation in the solutions was tracked over 42 days. The pigments formed were characterized as xanthylium structures regardless of the flavanol isomer present in the solution. The solutions supplied with Lactobacillus plantarum RM71 were oxidized at a slower rate, and consequently, its final color was less than that in the controls. The formation of both (þ)-catechin and (-)-epicatechin-derived xanthylium pigments was also delayed over time in the presence of the bacterium compared to their respective cell-free controls. The delaying effects provided by L. plantarum on the oxidative coloration and the generation of xanthylium-derived pigments were more pronounced for the (-)-epicatechin than for the (þ)-catechin model solutions. In view of these results and given that L. plantarum is naturally present in winemaking and generally recognized as a safe microorganism, the potential application of this bacterium as an antibrowning agent for wine is now opened

The potential to degrade 15 food phenolic acids was investigated for several Lactobacillus brevis strains isolated from different sources. All the strains analysed in this study showed a similar metabolism on phenolic acids. Among the cinnamic acids assayed, only p-coumaric, ferulic and caffeic acids were metabolized by the L. brevis strains. These acids were decarboxylated to produce their corresponding vinyl derivatives. Contrarily to the results previously reported on Lactobacillus plantarum, the L. brevis strains analysed in this study were unable to subsequently reduce or metabolize these vinyl derivatives. In L. brevis, vinyl phenol, vinyl catechol, and vinyl guaiacol were the final metabolic products from p-coumaric, caffeic or ferulic acids, respectively. From the benzoic acids analysed, and similarly to L. plantarum strains, only gallic and protocatechuic acids were modified by L. brevis strains. Both acids were decarboxylated to pyrogallol and catechol, respectively. Currently, the enzymes involved in the metabolism of phenolic acids in L. brevis remain uncharacterized, This work was supported by Grants AGL2005-00470, AGL2008- 01052, Consolider INGENIO 2010 CSD2007-00063 FUN-C-FOOD (CICYT), RM2008-00002 (INIA), and S-0505/AGR/000153 (CAM). We are grateful to M. V. Santamaría, H. Rodríguez, and J. A. Curiel were recipients of predoctoral fellowships from the I3P-CSIC Program and FPI-MEC, respectively. J. M. Landete was a recipient of a postdoctoral fellowship from the MEC.

In recent years, the exquisite stereoselectivity and high efficiency of carbohydrate-processing enzymes have been exploited for many biotechnological applications, including flavor enhancement in foods. In particular, much attention has been focused on the use of b-glucosidases for the enzymatic hydrolysis of flavorless glycoconjugates present in juices and wine beverages for the release aroma volatiles. With the aim to analyze a novel glycosidase with potential applications food industry we have produced and structurally characterized the Bgl glycosidase from the food lactic acid bacterium Lactobacillus plantarum. For that purpose, we have cloned and heterologously expressed the bgl gene (lp_3629) in Escherichia coli. The recombinant protein containing an amino terminal His6 tag (Bgl) has been produced in a soluble form. Purified recombinant enzyme shows galactosidase activity against 4-nitrophenyl b-D-galactopyranoside but not glucosidase activity. Analytical size-exclusion gel filtration chromatography reveals that Bgl behaves in solution as a mixture of monomeric and a high-molecular weight assembly. Purified Bgl has been crystallized by the hanging-drop vapor-diffusion method at 18 C. Diffraction data have been collected at ESRF to a resolution of 2.4 Å. The crystals belong to the space group C2 with unit-cell parameters a = 196.7, b = 191.7, c = 105.9, b = 102.7 . The structure refinement is in progress, JMM thanks the Ministerio de Educación y Ciencia for a research grant (BFU2007-67404/BMC) and ‘‘Factoría de Cristalización” Consolider- Ingenio 2010 in support of his research and the ESRF (Grenoble, France) for provision of synchrotron radiation facilities. This work was also supported by Grants AGL2008-01052 and CSD2007-00063 FUN-C-FOOD (MICINN), RM2008-00002 (INIA), and S-0505/AGR/000153 ALIBIRD (CAM). J.A. Curiel is a recipient of a FPI predoctoral fellowship from the MICINN.

Phenolic compounds are important constituents of food products of plant origin. These compounds are directly related to sensory characteristics of foods such as flavour, astringency, and colour. In addition, the presence of phenolic compounds on the diet is beneficial to health due to their chemopreventive activities against carcinogenesis and mutagenesis, mainly due to their antioxidant activities. Lactic acid bacteria (LAB) are autochthonous microbiota of raw vegetables. To get desirable properties on fermented plant-derived food products, LAB has to be adapted to the characteristics of the plant raw materials where phenolic compounds are abundant. Lactobacillus plantarum is the commercial starter most frequently used in the fermentation of food products of plant origin. However, scarce information is still available on the influence of phenolic compounds on the growth and viability of L. plantarum and other LAB species. Moreover, metabolic pathways of biosynthesis or degradation of phenolic compounds in LAB have not been completely described. Results obtained in L. plantarum showed that L. plantarum was able to degrade some food phenolic compounds giving compounds influencing food aroma as well as compounds presenting increased antioxidant activity. Recently, several L. plantarum proteins involved in the metabolism of phenolic compounds have been genetically and biochemically characterized. The aim of this review is to give a complete and updated overview of the current knowledge among LAB and food phenolics interaction, which could facilitate the possible application of selected bacteria or their enzymes in the elaboration of food products with improved characteristics., This work was supported by grants AGL2005-00470, AGL2008-01052, Consolider INGENIO 2010 CSD2007-00063 FUN-C-FOOD (CICYT), RM2008-00002 (INIA), and S-0505/AGR/000153 (CAM). H. Rodríguez and J. A. Curiel were recipients of predoctoral fellowships from the I3P-CSIC Program and FPI-MEC, respectively. J. M. Landete was a recipient of a postdoctoral fellowship from the MEC.

Chickpea seeds ( Cicer arietinum cv. Blanco lechoso ) were submitted to different bioprocesses; i) germination of seeds for 2 and 3 days, ii) natural fermentation of flour or cracked seeds, and iii) induced fermentation of flour or cracked seeds with Lactobacillus plantarum , in order to improve the antioxidant properties. Determination of vitamins C and E, reduced glutathione (GSH) and total phenolic compounds (TPC) were carried out. Antioxidant capacity was measured by SOD-like activity, peroxyl radical-trapping capacity (PRTC), lipid peroxidation inhibition (LPI) and trolox equivalent antioxidant capacity (TEAC). Vitamin C and E increased after germination, whilst fermentation caused a decrease in vitamin E. Both bioprocesses caused an increment in TPC content while GSH decreased. Germination increased SOD-like activity (47–41%), PRTC (16–55%) and TEAC (12–23%) and a slight inhibition of LPI was observed. After fermentation, SOD-like activity suffer a drastic reduction. The LPI decreased between 11.3 and 21.3%, while an increase of TEAC (29–57%) and PRTC (27–44%) was observed, except in flour natural fermented seeds where decreased the PRTC. TPC contributed highly to total antioxidant capacity (TEAC). Results indicated that with the bioprocesses studied the antioxidant properties of chickpea flours are enhanced and they can be used as desired ingredients for new functional food formulations.

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 determined, L. 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 appreciated

It was previously reported that cell cultures from Lactobacillus plantarum CECT 748T were able to decarboxylate phenolic acids, such as p-coumaric, m-coumaric, caffeic, ferulic, gallic, and protocatechuic acid. The p-coumaric acid decarboxylase (PDC) from this strain has been overexpressed and purified. This PDC differs at its C-terminal end when compared to the previously reported PDC from L. plantarum LPCHL2. Because the C-terminal region of PDC is involved in enzymatic activity, especially in substrate activity, it was decided to biochemically characterize the PDC from L. plantarum CECT 748T. Contrarily to L. plantarum LPCHL2 PDC, the recombinant PDC from L. plantarum CECT 748T is a heat-labile enzyme, showing optimal activity at 22 °C. This PDC is able to decarboxylate exclusively the hydroxycinnamic acids p-coumaric, caffeic, and ferulic acids. Kinetic analysis showed that the enzyme has a 14-fold higher KM value for p-coumaric and caffeic acids than for ferulic acid. PDC catalyzes the formation of the corresponding 4-vinyl derivatives (vinylphenol and vinylguaiacol) from p-coumaric and ferulic acids, respectively, which are valuable food additives that have been approved as flavoring agents. The biochemical characteristics showed by L. plantarum PDC should be taken into account for its potential use in the food-processing industry, The technical assistance of M. V. Santamaría is greatly appreciated.

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 substrate-inducible p-coumaric acid decarboxylase (PDC) from Lactobacillus plantarum has been overexpressed in Escherichia coli, purified and confirmed to possess decarboxylase activity. The recombinant His6-tagged enzyme was crystallized using the hanging-drop vapour-diffusion method from a solution containing 20%(w/v) PEG 4000, 12%(w/v) 2-propanol, 0.2 M sodium acetate, 0.1 M Tris–HCl pH 8.0 with 0.1 M barium chloride as an additive. Diffraction data were collected in-house to 2.04 A ° resolution. Crystals belonged to the tetragonal space group P43, with unit-cell parameters a = b = 43.15, c = 231.86 A ° . The estimated Matthews coefficient was 2.36 A ° 3 Da 1, corresponding to 48% solvent content, which is consistent with the presence of two protein molecules in the asymmetric unit. The structure of PDC has been determined by the molecular-replacement method. Currently, the structure of PDC complexed with substrate analogues is in progress, with the aim of elucidating the structural basis of the catalytic mechanism, JMM thanks the Ministerio de Educación y Ciencia for a research grant (BFU2004-01554/BMC; DGCYT) in support of his research. This work was also supported by grants AGL2005-00470 (CICYT) and S-0505/AGR000153 (CAM). HR is a recipient of a I3P predoctoral fellowship from the CSIC.

Cowpea (Vigna sinensis L. var. Carilla) flours obtained by fermentation with inoculum Lactobacillus plantarum (PF) or with the natural microorganisms present in the flour (NF) and subsequent heat treatment in an autoclave were prepared to study the effect of fermentation on the antioxidant vitamin content and on the antioxidant capacity. Bacterial counts and pH values, vitamins C and E, carotenoids, glutathione (GSH), superoxide dismutase-like activity (SOD-like activity), peroxyl radical-trapping capacity (PRTC), lipid peroxidation in unilamillar liposomes, and Trolox equivalent antioxidant capacity (TEAC) were evaluated in raw and processed cowpea flours. ç-Tocopherol and ä-tocopherol were found in raw cowpea, whereas vitamin C and carotenoids were not detected. An increase in the vitamin E activity was observed in PF, whereas vitamin C and carotenoids were not detected in fermented cowpea flours. Fermentation or heat treatment in an autoclave after fermentation produced processed cowpea flours with lower PRTC, glutathione content, and SOD-like activity than those of the raw seeds. However, those processes increased the capacity to inhibit the lipid peroxidation in unilamellar lipoposomes and TEAC. According to the results obtained in this study, the fermentation of cowpeas (naturally or with L. plantarum) and fermentation and subsequent heat treatment in an autoclave are good processes to obtain functional cowpea flours having higher antioxidant capacity than the raw legume

The authors are grateful to the Spanish Comisi´on Interministerial de Ciencia y Tecnolog´ia (AGL2000- 1609-C02) and also to the Comunidad Aut´onoma de Madrid for a predoctoral grant.

© 2015, American Society for Microbiology. Lactobacillus plantarum is the lactic acid bacterial species most frequently found in the fermentation of food products of plant origin on which phenolic compounds are abundant. L. plantarum strains showed great flexibility in their ability to adapt to different environments and growth substrates. Of 28 L. plantarum strains analyzed, only cultures from 7 strains were able to hydrolyze hydroxycinnamic esters, such as methyl ferulate or methyl caffeate. As revealed by PCR, only these seven strains possessed the est_1092 gene. When the est_1092 gene was introduced into L. plantarum WCFS1 or L. lactis MG1363, their cultures acquired the ability to degrade hydroxycinnamic esters. These results support the suggestion that Est_1092 is the enzyme responsible for the degradation of hydroxycinnamic esters on the L. plantarum strains analyzed. The Est_1092 protein was recombinantly produced and biochemically characterized. Surprisingly, Est_1092 was able to hydrolyze not only hydroxycinnamic esters, since all the phenolic esters assayed were hydrolyzed. Quantitative PCR experiments revealed that the expression of est_1092 was induced in the presence of methyl ferulate, an hydroxycinnamic ester, but was inhibited on methyl gallate, an hydroxybenzoic ester. As Est_1092 is an enzyme active on a broad range of phenolic esters, simultaneously possessing feruloyl esterase and tannase activities, its presence on some L. plantarum strains provides them with additional advantages to survive and grow on plant environments., This work was supported by grants AGL2011-22745 (MINECO) and RM2012-00004 (INIA). M. Esteban-Torres is the recipient of a JAE predoctoral fellowship from CSIC. J. M. Landete is the recipient of a Ramón y Cajal contract, and L. Santamaría is the recipient of an FPI fellowship from MINECO.

Mung beans (Vigna radiata [L] Wilczek) purchased from a Spanish company as >green soybeans>, showed a different phenolic composition than yellow soybeans (Glycine max cv. Merit). Isoflavones were predominant in yellow soybeans, whereas they were completely absent in the green seeds on which flavanones were predominant. In order to enhance their health benefits, both types of bean were subjected to technological processes, such as soaking and fermentation. Soaking increased malonyl glucoside isoflavone extraction in yellow beans and produced an increase in apigenin derivatives in the green beans. Lactobacillus plantarum CECT 748T fermentation produced an increase in the bioactivity of both beans since a conversion of glycosylated isoflavones into bioactive aglycones and an increase of the bioactive vitexin was observed in yellow and green beans, respectively. In spite of potential consumer confusion, since soybean and >green soybean> are different legumes, the health benefits of both beans were enhanced by lactic fermentation., This work was supported by grants RM2012-00004 (Instituto Nacional de Investigación Agraria y Alimentaría), AGL2008-01052, AGL2011-22745, Consolider INGENIO 2010 CSD2007-00063 FUN-C-FOOD (MINECO) y S2009/AGR-1469 (ALIBIRD) (Comunidad de Madrid).

© 2014 American Dairy Science Association. Lactobacillus plantarum is a lactic acid bacterium that can be found during cheese ripening. Lipolysis of milk triacylglycerols to free fatty acids during cheese ripening has fundamental consequences on cheese flavor. In the present study, the gene lp_1760, encoding a putative esterase or lipase, was cloned and expressed in Escherichia coli BL21 (DE3) and the overproduced Lp_1760 protein was biochemically characterized. Lp_1760 hydrolyzed p-nitrophenyl esters of fatty acids from C2 to C16, with a preference for p-nitrophenyl butyrate. On triglycerides, Lp_1760 showed higher activity on tributyrin than on triacetin. Although optimal conditions for activity were 45°C and pH 7, Lp_1760 retains activity under conditions commonly found during cheese making and ripening. The Lp_1760 showed more than 50% activity at 5°C and exhibited thermal stability at high temperatures. Enzymatic activity was strongly inhibited by sodium dodecyl sulfate and phenylmethylsulfonyl fluoride. The Lp_1760 tributyrin esterase showed high activity in the presence of NaCl, lactic acid, and calcium chloride. The results suggest that Lp_1760 might be a useful tributyrin esterase to be used in cheese manufacturing., This work was financially supported by grants AGL2011-22745 and BFU2010-17929 (MINECO), S2009/AGR/1469 (ALIBIRD) (Comunidad de Madrid), and RM2012-00004 (INIA). We are grateful to J.M. Barcenilla and M. V. Santamaría. M. Esteban-Torres is a recipient of a JAE predoctoral fellowship from the CSIC.

[Background] Herbivores have developed mechanisms to overcome adverse effects of dietary tannins through the presence of tannin-resistant bacteria. Tannin degradation is an unusual characteristic among bacteria. Streptococcus gallolyticus is a common tannin-degrader inhabitant of the gut of herbivores where plant tannins are abundant. The biochemical pathway for tannin degradation followed by S. gallolyticus implies the action of tannase and gallate decarboxylase enzymes to produce pyrogallol, as final product. From these proteins, only a tannase (TanBSg) has been characterized so far, remaining still unknown relevant proteins involved in the degradation of tannins., [Results] In addition to TanBSg, genome analysis of S. gallolyticus subsp. gallolyticus strains revealed the presence of an additional protein similar to tannases, TanASg (GALLO_0933). Interestingly, this analysis also indicated that only S. gallolyticus strains belonging to the subspecies “gallolyticus” possessed tannase copies. This observation was confirmed by PCR on representative strains from different subspecies. In S. gallolyticus subsp. gallolyticus the genes encoding gallate decarboxylase are clustered together and close to TanBSg, however, TanASg is not located in the vicinity of other genes involved in tannin metabolism. The expression of the genes enconding gallate decarboxylase and the two tannases was induced upon methyl gallate exposure. As TanBSg has been previously characterized, in this work the tannase activity of TanASg was demonstrated in presence of phenolic acid esters. TanASg showed optimum activity at pH 6.0 and 37°C. As compared to the tannin-degrader Lactobacillus plantarum strains, S. gallolyticus presented several advantages for tannin degradation. Most of the L. plantarum strains possessed only one tannase enzyme (TanBLp), whereas all the S. gallolytcius subsp. gallolyticus strains analyzed possesses both TanASg and TanBSg proteins. More interestingly, upon methyl gallate induction, only the tanB Lp gene was induced from the L. plantarum tannases; in contrast, both tannase genes were highly induced in S. gallolyticus. Finally, both S. gallolyticus tannase proteins presented higher activity than their L. plantarum counterparts., [Conclusions] The specific features showed by S. gallolyticus subsp. gallolyticus in relation to tannin degradation indicated that strains from this subspecies could be considered so far the best bacterial cellular factories for tannin degradation., This work was financially supported by grants AGL2011-22745, S2009/AGR-1469 (ALIBIRD) (Comunidad de Madrid), and RM2012-00004 (INIA). We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

In situ extractive fermentation was used to produce 4-vinyl derivatives from hydroxycinnamic acids extracted from corn cobs by recombinant Escherichia coli cells expressing Lactobacillus plantarum phenolic acid descarboxylase (PAD) gene. This microorganism mainly produced 4-vinylphenol (4VP) from p-coumaric acid (p-CA). In the first study , we observed that the concentrations of 4VP are higher than 1g/L which had a negative impact on decarboxylation of p-CA to 4VP by recombinant E. coli cells. Because of this, and in order to improve the downstream process, a two-phase aqueous-organic solvent system was developed. The results of the extractive fermentation indicated that it was possible to use hydrolyzates as aqueous phase to bioproduce 4VP, and recover simultaneously the product in the organic phase containing hexane. The detoxification of pre-treated corn cob alkaline hydrolyzate improved 4VP production up to 1003.5mg/L after 24h fermentation (QP=41.813mg/Lh). Additionally, preliminary experiments using cells immobilized in calcium alginate showed to be a good system for the biotransform of p-CA to 4VP in extractive fermentation, although the process hindered partially the recovery of 4VP in the organic phase. © 2014 Elsevier Inc., Spanish Ministry of Science and Innovation for the financial support of this work (CTQ2011-28967 and AGL2011-22745 projects), which has partial financial support from the FEDER funds of the European Union. J.M.S. is grateful for Postdoctoral fellowship (SFRH / BPD / 84440 / 2012) of Fundação para a Ciência e Tecnologia – FCT.

© 2014 Elsevier Ltd. During cheese ripening, the metabolic activity of microorganisms influences cheese flavour. Lactobacillus plantarum is a non-starter lactic acid bacterium that can be found during cheese ripening. An esterase-encoding gene from L. plantarum, lp_3505, was cloned and expressed in Escherichia coli BL21 (DE3). The biotechnologically produced Lp_3505 protein was purified as an active soluble form using His-tag affinity chromatography. The enzyme has an optimal pH and temperature of 6 and 5 °C, respectively. The enzyme showed remarkable stability at 20 °C, retaining more than 60% of its maximal activity after 20 h incubation at this temperature. In addition, NaCl concentrations lower than 20% increased esterase activity. The cold-activity and salt-tolerance exhibited by Lp_3505 indicated that this esterase could be a useful exogenous enzyme to be added for cheese ripening., This work was financially supported by grants AGL2011-22745 and BFU2010-17929 (MINECO), S2009/AGR-1469 (ALIBIRD) (Comunidad de Madrid), and RM2012-00004 (INIA)

Lactobacillus plantarum is a lactic acid bacteria that can be found in numerous fermented foods. Esterases from L. plantarum exert a fundamental role in food aroma. In the present study, the gene lp-2631 encoding a putative esterase was cloned and expressed in Escherichia coli BL21 (DE3) and the overproduced Lp-2631 protein has been biochemically characterized. Lp-2631 exhibited optimal esterase activity at 20 °C and more than 90% of maximal activity at 5 °C, being the first cold-active esterase described in a lactic acid bacteria. Lp-2631 exhibited 40% of its maximal activity after 2 h of incubation at 65 °C. Lp-2631 also showed marked activity in the presence of compounds commonly found in food fermentations, such as NaCl, ethanol, or lactic acid. The results suggest that Lp-2631 might be a useful esterase to be used in food fermentations. © 2014 American Chemical Society., This work was financially supported by grants AGL2011-22745 and BFU2010-17929 (MINECO), S2009/AGR-1469 (ALIBIRD) (Comunidad de Madrid), and RM2012-00004 (INIA). M. Esteban-Torres is a recipient of a JAE predoctoral fellowship from the CSIC.

The hydrolase fold is one of the most versatile structures in the protein realm according to the diversity of sequences adopting such a three-dimensional architecture. In the present study, we clarified the crystal structure of the carboxylesterase Cest-2923 from the lactic acid bacterium Lactobacillus plantarum WCFS1 refined to 2.1 Å resolution, determined its main biochemical characteristics and also carried out an analysis of its associative behaviour in solution. We found that the versatility of a canonical α/β hydrolase fold, the basic framework of the crystal structure of Cest-2923, also extends to its oligomeric behaviour in solution. Thus, we discovered that Cest-2923 exhibits a pH-dependent pleomorphic behaviour in solution involving monomers, canonical dimers and tetramers. Although, at neutral pH, the system is mainly shifted to dimeric species, under acidic conditions, tetrameric species predominate. Despite these tetramers resulting from the association of canonical dimers, as is commonly found in many other carboxylesterases from the hormone-sensitive lipase family, they can be defined as 'noncanonical' because they represent a different association mode. We identified this same type of tetramer in the closest relative of Cest-2923 that has been structurally characterized: the sugar hydrolase YeeB from Lactococcus lactis. The observed associative behaviour is consistent with the different crystallographic results for Cest-2923 from structural genomics consortia. Finally, the presence of sulfate or acetate molecules (depending on the crystal form analysed) in the close vicinity of the nucleophile Ser116 allows us to identify interactions with the putative oxyanion hole and deduce the existence of hydrolytic activity within Cest-2923 crystals. Structured digital abstract Cest-2923 and Cest-2923 bind by x-ray crystallography (1, 2)Cest-2923 and Cest-2923 bind by cosedimentation in solution (1, 2) Database The atomic coordinates and structure factors have been deposited in the Protein Data Bank with accession numbers: 4BZW for Cest-2923 from native crystals not soaked with substrates (P6322 space group); 4C01 for Cest-2923 from crystals soaked with phenyl acetate (C2 space group); 4BZZ for Cest-2923 from crystals soaked with isopropenyl acetate (P622 space group). The analysis of the carboxylesterase Cest-2923 from the lactic acid bacterium Lactobacillus plantarum WCFS1 reveals that the intrinsically structural versatility of a canonical α/β-hydrolase fold also extends to its oligomeric behaviour in solution. Hence, we found that Cest-2923, exhibits a complex, pH-dependent pleomorphic behaviour involving monomers, canonical dimers and noncanonical tetramers. © 2013 FEBS., Financial support received from the Ministerio de Economıa y Competitividad (BFU2010-17929/BMC), as well as from Factorıa de Cristalizacion (Consolider-Ingenio-2007) (J.M.M.), AGL2011-22745 (R.M.) and FUN-C-FOOD Consolider 25506 (R.M.), is greatly appreciated. Y.A., M.E.-T. and I.A. are recipients of fellowships: CSICCITMA, JAE Predoc (CSIC) and FPU (MEC), respectively.

This is an open-access article distributed under the terms of the Creative Commons Attribution License., [Background]:Owing to its antimicrobial properties dietary tannins may alter the functional efficacy of probiotic lactobacilli in the gastrointestinal (GI)-tract influencing their growth, viability and molecular adaptation to the intestinal environment. [Methods and Findings]:The effects of tannic acid on Lactobacillus plantarum WCFS1 were studied by in vitro growth monitoring and visualizing the morphological alteration on the cell wall using transmission electron microscopy. Growth upon tannic acid was characterized by dose-dependent reductions of initial viable counts and extended lag phases. Lag phase-cells growing upon 0.5 mM tannic acid were abnormally shaped and experienced disturbance on the cell wall such as roughness, occasional leakage and release of cell debris, but resumed growth later at tannic acid concentrations high as 2.5 mM. To gain insight on how the response to tannic acid influenced the molecular adaptation of L. plantarum to the GI-tract conditions, gene expression of selected biomarkers for GI-survival was assessed by RT-qPCR on cDNA templates synthetized from mRNA samples obtained from cells treated with 0.5 or 2 mM tannic acid. Tannic acid-dependent gene induction was confirmed for selected genes highly expressed in the gut or with confirmed roles in GI-survival. No differential expression was observed for the pbp2A gene, a biomarker negatively related with GI-survival. However PBP2A was not labeled by Bocillin FL, a fluorescent dye-labeled penicillin V derivative, in the presence of tannic acid which suggests for enhanced GI-survival reportedly associated with the inactivation of this function. [Conclusions]:Probiotic L. plantarum WCFS1 is able to overcome the toxic effects of tannic acid. This dietary constituent modulates molecular traits linked to the adaptation to intestinal environment in ways previously shown to enhance GI-survival. © 2013 Reverón et al., H. Rodriguez and J. A. Curiel acknowledge I3P (CSIC) or FPI (MICINN) PhD grants, respectively. The authors gratefully acknowledge the partial financial support of the CICYT grants AGL2008-001052, AGL2011-22745, Consolider INGENIO 2010 CSD2007-00063 FUN-C-FOOD, the CAM grant S2009/AGR-1469 (ALIBIRD) and the MINECO grant CTM2012-38222-C02-02.

Scope: To advance knowledge of the stress tolerance mechanisms of a probiotic Lactobacillus plantarum strain to dietary hydroxycinnamic acids and the role of gut commensal microorganisms in the bioactivation of polyphenols. Methods and results: To understand how gut commensal microorganisms tolerate toxicity of hydroxycinnamic acids and bioactivate these compounds, we used whole genome transcriptional profiling to characterize the response of a L. plantarum human isolate during challenge with p-coumaric acid (p-CA). The transcriptional profile reveals a massive induction of genes involved in stress resistance and detoxification-related functions and a global shutdown of growth-associated processes. A specific oxidative stress response, including a large reshape of nitrogen metabolism toward methionine production, was induced, probably to counteract a p-CA-induced oxidative protein stress. The transcriptional datasets revealed overlapping behaviors with the response of L. plantarum to the gut environment. Conclusion: Contact with p-CA triggers responses that would be potentially beneficial for the intestinal function such as detoxification of dietary hydroxycinnamic acids and induction of a marked antioxidant response. Elicited responses indicated that contact with p-CA could provide preparedness to L. plantarum for adaptation to the gut environment. This knowledge facilitates the way to design methods to improve probiotic cell survival in this habitat. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim., This work was supported by grants AGL2008–01052, AGL2011–22745, and Consolider INGENIO 2010 CSD2007–00063 FUN-C-FOOD (CICYT), and S2009/AGR-1469 (ALIBIRD) (CAM).

Q88Y25_Lacpl is an esterase produced by the lactic acid bacterium Lactobacillus plantarum WCFS1 that shows amino-acid sequence similarity to carboxylesterases from the hormone-sensitive lipase family, in particular the AFEST esterase from the archaeon Archaeoglobus fulgidus and the hyperthermophilic esterase EstEI isolated from a metagenomic library. N-terminally His6-tagged Q88Y25_Lacpl has been overexpressed in Escherichia coli BL21 (DE3) cells, purified and crystallized at 291 K using the hanging-drop vapour-diffusion method. Mass spectrometry was used to determine the purity and homogeneity of the enzyme. Crystals of His6-tagged Q88Y25_Lacpl were prepared in a solution containing 2.8 M sodium acetate trihydrate pH 7.0. X-ray diffraction data were collected to 2.24 A ¿ resolution on beamline ID29 at the ESRF. The apparent crystal point group was 422; however, initial global analysis of the intensity statistics (data processed with high symmetry in space group I422) and subsequent tests on data processed with low symmetry (space group I4) showed that the crystals were almost perfectly merohedrally twinned. Most probably, the true space group is I4, with unit-cell parameters a = 169.05, b = 169.05, c = 183.62 A, JMM and RM thank the Ministerio de Educación y Ciencia for research grants (BFU2010-17929/BMC, AGL2008-01052 and AGL2011-22745; DGCYT), ‘Factoría de Cristalización’ (CSD2006-00015) and FUN-C-FOOD (CSD2007-00063) Consolider-Ingenio 2010 and ALIBIRD S2009/AGR-1469 (CAM) and RM2008-00002 (INIA) for support of their research.

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.