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Gut microbiota is a constant source of antigens and stimuli to which the resident immune system has developed tolerance. However, the mechanisms by which mononuclear phagocytes, specifically monocytes/macrophages, cope with these usually pro-inflammatory signals are poorly understood. Here, we show that innate immune memory promotes anti-inflammatory homeostasis, using as model strains of the commensal bacterium Lactiplantibacillus plantarum. Priming of monocytes/macrophages with bacteria, especially in its live form, enhances bacterial intracellular survival and decreases the release of pro-inflammatory signals to the environment, with lower production of TNF and higher levels of IL-10. Analysis of the transcriptomic landscape of these cells shows downregulation of pathways associated with the production of reactive oxygen species (ROS) and the release of cytokines, chemokines and antimicrobial peptides. Indeed, the induction of ROS prevents memory-induced bacterial survival. In addition, there is a dysregulation in gene expression of several metabolic pathways leading to decreased glycolytic and respiratory rates in memory cells. These data support commensal microbe-specific metabolic changes in innate immune memory cells that might contribute to homeostasis in the gut., Supported by grants from the Spanish Ministry of Science, Innovation and Universities (MCIU) co-financed with FEDER funds (RTI2018-096494-B-100 to JA; BFU2016-76872-R to EB; AGL2017-86757-R to LA; SAF2015-73549-JIN to HR; SAF2016–77433-R and PID2019-110240RB-I00 to RPR). AP is supported by a Postdoctoral Fellowship from the Basque Government. DB and TMM are recipients of MCIU FPI fellowships. APC is a recipient of a fellowship from the University of the Basque Country. LA and RPR are supported by the Ramon y Cajal program from the Spanish Ministry of Economy and Competitiveness. We thank the MCIU for the Severo Ochoa Excellence accreditation (SEV-2016-0644), the Basque Department of Industry, Tourism and Trade (Etortek and Elkartek programs) and the Innovation Technology Department of the Bizkaia Province. This work was further supported by grants from the Jesús de Gangoiti Barrera Foundation.

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).

Colorectal cancer pathogenesis and progression isassociated with the presence of Fusobacteriumnucleatum and the reduction of acetylated deriva-tives of spermidine, as well as dietary componentssuch as tannin-rich foods. We show that a new tan-nase orthologue of F. nucleatum (TanBFnn) has sig-nicant structural differences with its Lactobacillusplantarum counterpart affecting the ap covering theactive site and the accessibility of substrates. Crys-tallographic and molecular dynamics analysisrevealed binding of polyamines to a small cavity thatconnects the active site with the bulk solvent whichinteract with catalytically indispensable residues. Asa result, spermidine and its derivatives, particularlyN8-acetylated spermidine, inhibit the hydrolytic activ-ity of TanBFnnand increase the toxicity of gallotan-nins to F. nucleatum. Our results support a model inwhich the balance between the detoxicant activity ofTanBFnnand the presence of metabolic inhibitorscan dictate either conducive or unfavourable condi-tions for the survival of F. nucleatum., Supported by grants from the Spanish Ministry of Science and Innovation (MCI) cofinanced with FEDER funds (SAF2015‐65327‐R and RTI2018‐096494‐B‐100 to JA; AGL2017‐86757‐R to LA, SAF2015‐73549‐JIN to HR; RTI2018‐099592‐B‐C22 to GJO) and the Mizutani Foundation for Glycoscience (200077 to GJO). LA and GJO are supported by the Ramon y Cajal program (RYC‐2013‐13666 and RYC‐2013‐14706 respectively). JTC and AP are the recipients postdoctoral fellowships from the Basque Government. DB is the recipient of a MCI FPI fellowship. APC is the recipient of a fellowship from the University of the Basque Country. We thank the MCI for the Severo Ochoa Excellence accreditation (SEV‐2016‐0644) and the Basque Department of Industry, Tourism and Trade (Etortek and Elkartek programs). JMM thanks the ALBA synchrotron for providing access time to the BL‐13 XALOC beamline. This work is supported by grants from the Jesús de Gangoiti Barrera Foundation

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.

Different Pd-complexes containing orthometallated push–pull oxazolones were inserted by supramolecular Pd-amino acid coordination on two genetically engineered modified variants of the thermoalkalophilic Geobacillus thermocatenolatus lipase (GTL). Pd-lipase conjugation was performed on the solid phase in the previously immobilized form of GTL under mild conditions, and soluble conjugated Pd-GTL complexes were obtained by simply desorbing by washing with an acetonitrile aqueous solution. Three different Pd complexes were incorporated into two different genetically modified enzyme variants, one containing all the natural cysteine residues changed to serine residues, and another variant including an additional Cys mutation directly in the catalytic serine (Ser114Cys). The new Pd–enzyme conjugates were fluorescent even at ppm concentrations, while under the same conditions free Pd complexes did not show fluorescence at all. The Pd conjugation with the enzyme extremely increases the catalytic profile of the corresponding Pd complex from 200 to almost 1000-fold in the hydrogenation of arenes in aqueous media, achieving in the case of GTL conjugated with orthopalladated 4a an outstanding TOF value of 27 428 min−1. Also the applicability of GTL-C114 conjugated with orthopalladated 4b in a site-selective C–H activation reaction under mild conditions has been demonstrated. Therefore, the Pd incorporation into the enzyme produces a highly stable conjugate, and improves remarkably the catalytic activity and selectivity, as well as the fluorescence intensity, of the Pd complexes., We thank the Spanish National Research Council (CSIC, Spain), the Aragón Government (Spain, Project LMP144_18: Programa Operativo FEDER Aragón 2014-2020, “Construyendo Europa desde Aragón” and research group Aminoácidos y Péptidos E19_20R) and the Spanish Government (Ministerio de Ciencia e Innovación) (Projects No. AGL2017-84614-C2-2-R and PID2019-106394GB-I00) for funding. Authors thank the European Cooperation in Science and Technology (COST) program under CA15106 grant (CHAOS: CH Activation in Organic Synthesis). A. P. is grateful for the financial support of the Romanian Ministry of Education and Research through the grant PN-III-P1-1.1-MC-2018-2580., We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

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).

Three novel glycosidases produced from Lactobacillus plantarum, so called Lp_0440, Lp_2777, and Lp_3525, were isolated and overexpressed on Escherichia coli containing a His-tag for specific purification. Their specific activity was evaluated against the hydrolysis of p-nitrophenylglycosides and p-nitrophenyl-6-phosphate glycosides (glucose and galactose) at pH 7. All three were modified with hyaluronic acid (HA) following two strategies: A simple coating by direct incubation at alkaline pH or direct chemical modification at pH 6.8 through preactivation of HA with carbodiimide (EDC) and N-hydroxysuccinimide (NHS) at pH 4.8. The modifications exhibited important effect on enzyme activity and specificity against different glycopyranosides in the three cases. Physical modification showed a radical decrease in specific activity on all glycosidases, without any significant change in enzyme specificity toward monosaccharide (glucose or galactose) or glycoside (C-6 position free or phosphorylated). However, the surface covalent modification of the enzymes showed very interesting results. The glycosidase Lp_0440 showed low glycoside specificity at 25 &deg, C, showing the same activity against p-nitrophenyl-glucopyranoside (pNP-Glu) or p-nitrophenyl-6-phosphate glucopyranoside (pNP-6P-Glu). However, the conjugated cHA-Lp_0440 showed a clear increase in the specificity towards the pNP-Glu and no activity against pNP-6P-Glu. The other two glycosidases (Lp_2777 and Lp_3525) showed high specificity towards pNP-6P-glycosides, especially to the glucose derivative. The HA covalent modification of Lp_3525 (cHA-Lp_3525) generated an enzyme completely specific against the pNP-6P-Glu (phosphoglycosidase) maintaining more than 80% of the activity after chemical modification. When the temperature was increased, an alteration of selectivity was observed. Lp_0440 and cHA-Lp_0440 only showed activity against p-nitrophenyl-galactopyranoside (pNP-Gal) at 40 &deg, C, higher than at 25 &deg, C in the case of the conjugated enzyme.

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).

Tannin acyl hydrolases, also known as tannases, are a group of enzymes critical for the transformation of tannins. The study of these enzymes, which initially evolved in different organisms to detoxify and/or use these plant metabolites, has nowadays become relevant in microbial enzymology research due to their relevant role in food tannin transformation. Microorganisms, particularly bacteria, are major sources of tannase. Cloning and heterologous expression of bacterial tannase genes and structural studies have been performed in the last few years. However, a systematic compilation of the information related to all recombinant tannases, their classification, and characteristics is missing. In this review, we explore the diversity of heterologously produced bacterial tannases, describing their substrate specificity and biochemical characterization. Moreover, a new classification based on sequence similarity analysis is proposed. Finally, putative tannases have been identified in silico for each group of tannases taking advantage of the use of the "tannase" distinctive features previously proposed., This work was financially supported by grants AGL2014-52911-R and SAF2015-73549-JIN from the Spanish Ministry of Economy and Competitiveness/FEDR-MINEICO.

A novel heterogeneous enzyme-palladium (Pd) (0) nanoparticles (PdNPs) bionanohybrid has been synthesized by an efficient, green, and straightforward methodology. A designed Geobacillus thermocatenulatus lipase (GTL) variant genetically and then chemically modified by the introduction of a tailor-made cysteine-containing complementary peptide- was used as the stabilizing and reducing agent for the in situ formation of ultra-small PdNPs nanoparticles embedded on the protein structure. This bionanohybrid was an excellent catalyst in the synthesis of trans-ethyl cinnamate by Heck reaction at 65 ◦C. It showed the best catalytic performance in dimethylformamide (DMF) containing 10–25% of water as a solvent but was also able to catalyze the reaction in pure DMF or with a higher amount of water as co-solvent. The recyclability and stability were excellent, maintaining more than 90% of catalytic activity after five cycles of use., This work has been sponsored by the Spanish Government (Project AGL2017-84614-C2-2-R) and CSIC Project CSIC-PIE 201880E011).

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., This work was supported by MINECO grant AGL2014-52911-R (AEI/FEDER, UE). L.S. is a recipient of a FPI Fellowship from the MINECO, and M.E.-T. is a recipient of an Irish Research Council postdoctoral grant (grant GOIPD/2017/1302)

[Background]: Tannases are tannin-degrading enzymes that have been described in fungi and bacteria as an adaptative mechanism to overcome the stress conditions associated with the presence of these phenolic compounds. [Results]: We have identified and expressed in E. coli a tannase from the oral microbiota member Fusobacterium nucleatum subs. polymorphum (TanBFnp). TanBFnp is the first tannase identified in an oral pathogen. Sequence analyses revealed that it is closely related to other bacterial tannases. The enzyme exhibits biochemical properties that make it an interesting target for industrial use. TanBFnp has one of the highest specific activities of all bacterial tannases described to date and shows optimal biochemical properties such as a high thermal stability: the enzyme keeps 100% of its activity after prolonged incubations at different temperatures up to 45 °C. TanBFnp also shows a wide temperature range of activity, maintaining above 80% of its maximum activity between 22 and 55 °C. The use of a panel of 27 esters of phenolic acids demonstrated activity of TanBFnp only against esters of gallic and protocatechuic acid, including tannic acid, gallocatechin gallate and epigallocatechin gallate. Overall, TanBFnp possesses biochemical properties that make the enzyme potentially useful in biotechnological applications. [Conclusions]: We have identified and characterized a metabolic enzyme from the oral pathogen Fusobacterium nucleatum subsp. polymorphum. The biochemical properties of TanBFnp suggest that it has a major role in the breakdown of complex food tannins during oral processing. Our results also provide some clues regarding its possible participation on bacterial survival in the oral cavity. Furthermore, the characteristics of this enzyme make it of potential interest for industrial use., Supported by a Grant from the Spanish Ministry of Economy and Com petitiveness/FEDER—MINECO—to H.R. (SAF2015-73549-JIN) and R.M. (AGL2014-52911-R). We thank the MINECO for the Severo Ochoa Excellence accreditation (SEV2016-0644), the Basque Department of Industry, Tourism and Trade (Etortek and Elkartek programs), the Innovation Technology Department of the Bizkaia Province and the CIBERehd network. D.B. is the recipient of a fellowship for Formación de Personal Investigador from MINECO

Efficient enzymatic synthesis of lactosyl-oligofructosides (LFOS) with a degree of polymerization from 4 to 8 was achieved in the presence of sucrose:lactosucrose and sucrose:lactose mixtures by transfructosylation reaction. The main synthesized LFOS which consist of β-2,1-linked fructose to lactosucrose: β-d-galactopyranosyl-(1 → 4)-α-d-glucopyranosyl-[(1 → 2)-β-d-fructofuranosyl]n-(1 → 2)-β-d-fructofuranoside (where n refers to the number of transferred fructose moieties) was structurally characterized by nuclear magnetic resonance (NMR). The maximum formation of LFOS was 81% (in weight with respect to the initial amount of lactosucrose) and was obtained after 24 h of transfructosylation reaction based on sucrose:lactosucrose (250 g L− 1 each) catalyzed by an inulosucrase from Lactobacillus gasseri DSM 20604 (IS). The production of LFOS in the presence of sucrose:lactose mixtures required a previous high-yield lactosucrose synthesis step catalyzed by using a levansucrase from Bacillus subtilis CECT 39 (LS) before the inulosucrase-catalyzed reaction. This novel one-pot bi-enzymatic system led to the synthesis of about 22% LFOS in weight, with respect to the initial amount of lactose (250 g L− 1). The results revealed a high specificity for the substrate involved in the inulosucrase-catalyzed reaction given that, although lactosucrose (O-β-d-galactopyranosyl-(1 → 4)-O-α-d-glucopyranosyl-(1 → 2)-β-d-fructofuranoside) acted as a strong acceptor of β-2,1-linked fructose, lactose (β-d-galactopyranosyl-(1 → 4)-α-d-glucose) was found to be an extremely weak acceptor., This work has been financed by project AGL2011-27884 from the Ministerio de Ciencia e Innovación. M. Díez-Municio is supported by CSIC through JAE-Pre Programme co-funded by ESF. M. Herrero thanks MICINN for his “Ramón y Cajal” contract.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license.-- This article belongs to the Section Natural Products., A novel β-galactosidase from Lactobacillus plantarum (LPG) was over-expressed in E. coli and purified via a single chromatographic step by using lowly activated IMAC (immobilized metal for affinity chromatography) supports. The pure enzyme exhibited a high hydrolytic activity of 491 IU/mL towards o-nitrophenyl β-D-galactopyranoside. This value was conserved in the presence of different divalent cations and was quite resistant to the inhibition effects of different carbohydrates. The pure multimeric enzyme was stabilized by multipoint and multisubunit covalent attachment on glyoxyl-agarose. The glyoxyl-LPG immobilized preparation was over 20-fold more stable than the soluble enzyme or the one-point CNBr-LPG immobilized preparation at 50°C. This β-galactosidase was successfully used in the hydrolysis of lactose and lactulose and formation of different oligosaccharides was detected. High production of galacto-oligosaccharides (35%) and oligosaccharides derived from lactulose (30%) was found and, for the first time, a new oligosaccharide derived from lactulose, tentatively identified as 3′-galactosyl lactulose, has been described., This work has been sponsored by Consolider INGENIO 2010 CSD2007-00063 FUNC-FOOD(CICYT), the Spanish Ministry of Science and Innovation (Project CTQ2009-07568), CSIC(Intramural Project 200980I133) and S2009/AGR-1469 (ALIBIRD) (CAM)., We acknowledge the support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

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.

Tailor-made peptides were investigated for site-specific tag labeling of Geobacillus thermocatenulatus lipase (GTL). GTL was first genetically modified by introducing a unique cysteine on the lid site of the enzyme to produce two variants (GTLσ-A193C and GTLσ-S196C). Chemical modification was performed by using a small library of cysteine-containing peptides. The synthesized peptide–lipase biocatalysts were highly stable, more active, more specific, and more selective toward different substrates than unmodified GTL. Very high enzyme thermostability of GTLσ-A193C modified with peptides Ac-Cys-Phe-Gly-Phe-Gly-Phe-CONH (1) and Ac-Cys-Phe-Phe-CONH (2) (>95 % activity after 24 h at 60 °C) was observed. The incorporation of 1 and 2 in GTLσ-S196C improved its catalytic activity in the hydrolysis of p-nitrophenyl butyrate by factors of three and greater than five, respectively. The specificity for short-chain versus long-chain esters was also strongly improved. The diacylglycerol activity of GTLσ-S196C was enhanced more than tenfold by the incorporation of 1 and more than threefold by modification of this variant with Ac-Cys-(Arg)-CONH (6) in the hydrolysis of 1-stearoyl-2-arachidonoyl-sn-glycerol. The enantioselectivity of GTLσ-S196C increased for all formed bioconjugates, and the GTLσ-S196C–1 conjugate was the most active and selective in the hydrolysis of dimethylphenyl glutarate at pH 7 (72 % ee), also showing an inversion in the enzyme enantiopreference., This work was sponsored by the Spanish National Research Council (CSIC). We thank the Ramon Areces Foundation for financial support.

© 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.).

This work addresses the high-yield and fast enzymatic production of theanderose, a naturally occurring carbohydrate, also known as isomaltosucrose, whose chemical structure determined by NMR is α-d-glucopyranosyl-(1 → 6)-α-d-glucopyranosyl-(1 → 2)-ß-d-fructofuranose. The ability of isomaltose to act as an acceptor in the Bacillus subtilis CECT 39 levansucrase-catalyzed transfructosylation reaction to efficiently produce theanderose in the presence of sucrose as a donor is described by using four different sucrose:isomaltose concentration ratios. The maximum theanderose concentration ranged from 122.4 to 130.4 g L-1, was obtained after only 1 h and at a moderate temperature (37 °C), leading to high productivity (109.7-130.4 g L-1h-1) and yield (up to 37.3%) values. The enzymatic synthesis was highly regiospecific, since no other detectable acceptor reaction products were formed. The development of efficient and cost-effective procedures for the biosynthesis of unexplored but appealing oligosaccharides as potential sweeteners, such as theanderose, could help to expand its potential applications which are currently limited by their low availability., This work has been funded by Optibiotix Health plc. L.R-A. thanks the Spanish Research Council (CSIC) and the Spanish Ministry of Economy and Competitiveness for a Juan de la Cierva contract.

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).

The occurrence of in vitro production of biogenic amines in bacteria isolated from ice-preserved sardine and mackerel was studied. Biogenic amine production was investigated by means of amino acid decarboxylation by growth on decarboxylase differential medium, biogenic amine detection by thinlayer chromatography (TLC) and decarboxylase gene detection by PCR. Decarboxylase medium overestimate the number of biogenic amine-producer strains, as the production of amine was confirmed by TLC in only five out the 17 presumptive strains. On the producer strains, PCR was used to confirm the presence of the genes encoding the amino acid decarboxylase responsible for the synthesis of these amines. Moreover, biogenic amine-producer bacteria were molecularly identified by sequencing their 16S rRNA. Form sardine, enterobacteria producing simultaneously several biogenic amines were isolated. A Kluyvera intermedia strain producing histamine, putrescine and cadaverine, and an Enterobacter asburiae strain producing only the diamines cadaverine and putrescine were identified. From mackerel, lactic acid bacteria from the Enterococcus durans species producing tyramine were isolated. This study constitutes the first description of the presence on these putatively harmful species on ice-preserved sardine and mackerel., This work was supported by grants RM2008-00002 (Instituto Nacional de Investigación Agraria y Alimentaria), AGL2008-01052, Consolider INGENIO 2010 CSD2007-00063 FUN-C-FOOD (Comisión Interministerial de Ciencia y Tecnología), S2009/AGR-1469 (ALIBIRD) (Comunidad de Madrid), Tunisian Ministry of High Education and Scientific Research and Programa de Cooperación Interuniversitaria e Investigación Científica (AECI), Ministerio de Asuntos Exteriores y Cooperación, Spain (B/019335/08). We are grateful to M. V. Santamaría and J. M. Barcenilla. J. A. Curiel and G. Landeta are recipients of predoctoral fellowships from the I3P-CSIC and CONACyT, respectively.

The ability to degrade ochratoxin A was studied in different bacteria with a well-known capacity to transform aromatic compounds. Strains belonging to Rhodococcus, Pseudomonas, and Brevibacterium genera were grown in liquid synthetic culture medium containing ochratoxin A. Brevibacterium spp. strains showed 100% degradation of ochratoxin A.Ochratoxin ¿ was detected and identified by high-performance liquid chromatographymass spectrometry (HPLC-MS) as a degradation product in the cellfree supernatants. The degradation of ochratoxin A is of public concern for food and environmental safety, because it could contribute to the development of new biological ochratoxin A detoxification systems in foodstuffs. In this study, the degradation of ochratoxin A by bacteria belonging to the food chain was demonstrated for the first time., This work was supported by the “ASER” grant (D.M 16101/7301/08) from the Italian Ministero delle Politiche Agricole Alimentari e Forestali and by grants RM2008-00002 (Instituto Nacional de Investigación Agraria y Alimentaria), AGL2008-01052, Consolider INGENIO 2010 CSD2007-00063 FUN-C-FOOD (Comisión Interministerial de Ciencia y Tecnología), and S2009/AGR-1469 (ALIBIRD) (Comunidad de Madrid).

Lipase from Geobacillus thermocatenulatus (BTL2) was immobilized in two different matrixes. In one derivative, the enzyme was immobilized on agarose activated with cyanogen bromide (CNBr-BTL2) via its most reactive superficial amino group, whereas the other derivative was covalently immobilized on glyoxyl agarose supports (Gx-BTL2). The latter immobilization protocol leads to intense multipoint covalent attachment between the lysine richest region of enzyme and the glyoxyl groups on the support surface. The resulted solid derivatives were unfolded by incubation under high concentrations of guanidine and then resuspended in aqueous media under different experimental conditions. In both CNBr-BTL2 and Gx-BTL2 derivatives, the oxidation of Cys residues during the unfolding/refolding processes led to inefficient folding for the enzyme because only 25–30% of its initial activity was recovered after 3 h in refolding conditions. Dithiothreitol (DTT), a very mild reducing agent, prevented Cys oxidation during the unfolding/refolding process, greatly improving activity recovery in the refolded forms. In parallel, other variables such as pH, buffer composition and the presence of polymers and other additives, had different effects on refolding efficiencies and refolding rates for both derivatives. In the case of solid derivatives of BTL2 immobilized on CNBr-agarose, the surface's chemistry was crucial to guarantee an optimal protein refolding. In this way, uncharged protein vicinities resulted in better refolding efficiencies than those charged ones.

In this work, we analyzed at high resolution the sugar-binding mode of the recombinant N-terminal ricin-B domain of the hemolytic protein LSLa (LSL150) from the mushroom Laetiporus sulphureus and also provide functional in vitro evidence suggesting that, together with its putative receptor-binding role, this module may also increase the solubility of its membrane pore-forming partner. We first demonstrate that recombinant LSL150 behaves as an autonomous folding unit and an active lectin. We have determined its crystal structure at 1.47 Å resolution and also that of the [LSL150:(lactose)β, γ)] binary complex at 1.67 Å resolution. This complex reveals two lactose molecules bound to the β and γ sites of LSL150, respectively. Isothermal titration calorimetry indicates that LSL150 binds two lactoses in solution with highly different affinities. Also, we test the working hypothesis that LSL150 exhibits in vivo properties typical of solubility tags. With this aim, we have fused an engineered version of LSL150 (LSLt) to the N-terminal end of various recombinant proteins. All the designed LSL150-tagged fusion proteins were successfully produced at high yield, and furthermore, the target proteins were purified by a straightforward affinity procedure on agarose-based matrices due to the excellent properties of LSL150 as an affinity tag. An optimized protocol for target protein purification was devised, which involved removal of the LSL150 tag through in-column cleavage of the fusion proteins with His6-tagged TEV endoprotease. These results permitted to set up a novel, lectin-based system for production and purification of recombinant proteins in E. coli cells with attractive biotechnological applications., Financial support from the Ministerio de Ciencia e Innovación (BFU2010-17929/BMC; BFU2009-10052) and the Factoría de Cristalización (Consolider-Ingenio-2007), and CIBER de Enfermedades Respiratorias (CIBERES), an initiative of Instituto de Salud Carlos III, is greatly appreciated.

Staphylococcus warneri strain EX17 produces three lipases with different molecular weights of 28, 30, and 45 kDa. The 45 kDa fraction (SWL-45) has been purified from crude protein extracts by one chromatographic step based on the selective adsorption of this lipase by interfacial activation on different hydrophobic supports at low ionic strength. The adsorption of SWL-45 on octyl-Sepharose increased the enzyme activity by 60%, but the other lipases were also adsorbed on this support. Using butyl-Toyopearl, which is a lesser hydrophobic support, the purification factor was close to 20, and the only protein band detected on the sodium dodecyl sulfate-polyacrylamide electrophoresis analysis gel was that corresponding to the SWL-45, which could be easily desorbed from the support by incubation with triton X-100, producing a purified enzyme. SWL-45 was immobilized under very mild conditions on cyanogen bromide Sepharose, showing similar activities and stability as for its soluble form but without intermolecular interaction. The effects of different detergents over the activity of the immobilized SWL-45 were analyzed, which was hyperactivated by factors of 1.3 and 2.5 with 0.01% Tween 80 and 0.1% Triton X-100, respectively, while ionic detergents produced detrimental effects on the enzyme activity even at very low concentrations. Optimal reaction conditions and the effect of other additives on the enzyme activity were also investigated.

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.

The activity of a lipase from Geobacillus thermocatenulatus (BTL2) can be greatly improved by site-directed chemical modification of a single external Cys64. This residue is placed in the proximity of the region where the lid is allocated when the lipase exhibits its open and active form. Thiol group of Cys64 was modified by thiol-disulfide exchange with pyridyldisulfide poly-aminated-dextrans or mono-carboxylated-polyethyleneglycol. The modification was performed on the covalently immobilized lipase on CNBr-agarose or glyoxyl-agarose. The activity of modified derivatives was strongly dependent on the immobilized preparation, the polymer used and the substrate assayed. For example, the modification with PEG-COOH of BTL2 immobilized on glyoxyl-agarose increased 5-fold the enzyme activity towards the hydrolysis of 2-O-butyryl-2-phenylacetic acid. However, the modification with 3-(2-pyridyldithio)-propionyl-dextran-NH2 reduced the activity to 40%. The fact that the modified enzymes can be inhibited by an irreversible inhibitor much more rapidly than the unmodified ones suggested that the main effect of the modification is to somehow stabilize the open form of the lipase. © 2009 Elsevier Ltd. All rights reserved.

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.

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.

Enterococcus faecium RM58 produces beta-phenylethylamine and tyramine. A gene from Ent. faecium RM58 coding for a 625 amino-acid residues protein that shows 85% identity to Enterococcus faecalis tyrosine decarboxylase has been expressed in Escherichia coli, resulting in L-phenylalanine and L-tyrosine decarboxylase activities. Both activities were lost when a truncated protein lacking 84 amino acids at its C-terminus was expressed in E. coli. This study constitutes the first genetic characterization of a bacterial protein having L-phenylalanine decarboxylase activity and solves a long-standing question regarding the specificity of tyrosine decarboxylases in enterococci.

© 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).

The microorganisms in the human gastrointestinal tract have a profound influence on the transformation of food into metabolites which can impact human health. Gallic acid (GA) and pyrogallol (PG) are bioactive compounds displaying diverse biological properties, including carcinogenic inhibiting activities. However, its concentration in fruits and vegetables is generally low. These metabolites can be also generated as final products of tannin metabolism by microbes endowed with tannase, which opens up the possibility of their anti-cancer potential being increased. Patients with colorectal cancer (CRC) display an imbalanced gut microbiota respect to healthy population. The recent use of next generation sequencing technologies has greatly improved knowledge of the identity of bacterial species that colonize non-tumorous and tumorous tissues of CRC patients. This information provides a unique opportunity to shed light on the role played by gut microorganisms in the different stages of this disease. We here review the recently published gut microbiome associated to CRC patients and highlight tannase as an underlying gene function of bacterial species that selectively colonize tumorous tissues, but not adjacent non-malignant tissues. Given the anti-carcinogenic roles of GA and PG produced by gut tannin-degrading bacteria, we provide an overview of the possible consequences of this intriguing coincidence for CRC development., This work was supported by grant AGL2011-22745.

[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).

The gene in the locus GALLO-1609 from Streptococcus gallolyticus UCN34 was cloned and expressed as an active protein in Escherichia coli BL21 (DE3). The protein was named TanSg1 since it shows similarity to bacterial tannases previously described. The recombinant strain produced His-tagged TanSg1 which was purified by affinity chromatography. Purified TanSg1 protein showed tannase activity, having a specific activity of 577 U/mg which is 41 % higher than the activity of Lactobacillus plantarum tannase. Remarkably, TanSg1 displayed optimum catalytic activity at pH 6-8 and 50-70°C and showed high stability over a broad range of temperatures. It retained 25 % of its relative activity after prolonged incubation at 45°C. The specific activity of TanSg1 is enhanced by the divalent cation Ca2+ and is dramatically reduced by Zn2+ and Hg2+. The enzyme was highly specific for gallate and protocatechuate esters and showed no catalytic activity against other phenolic esters. The protein TanSg1 hydrolyzes efficiently tannic acid, a complex and polymeric gallotanin, allowing its complete conversion to gallic acid, a potent antioxidant. From its biochemical properties, TanSg1 is a tannase with potential industrial interest regarding the biodegradation of tannin waste or its bioconversion into biologically active products. © 2014 Springer-Verlag., This work was financially supported by grants AGL2011-22745, S2009/AGR-1469 (ALIBIRD) (Comunidad de Madrid), and RM2012-00004 (INIA). We are grateful to M. V. Santamaría for her technical assistance. N. Jiménez is a recipient of a FPI fellowship from the MINECO.

The histidine decarboxylase gene cluster of Morganella morganii DSM30146T was sequenced, and four open reading frames, named hdcT1, hdc, hdcT2, and hisRS were identified. Two putative histidine/histamine antiporters (hdcT1 and hdcT2) were located upstream and downstream the hdc gene, codifying a pyridoxal-P dependent histidine decarboxylase, and followed by hisRS gene encoding a histidyl-tRNA synthetase. This organization was comparable with the gene cluster of other known Gram negative bacteria, particularly with that of Klebsiella oxytoca. Recombinant Escherichia coli strains harboring plasmids carrying the M. morganii hdc gene were shown to overproduce histidine decarboxylase, after IPTG induction at 37 C for 4 h. Quantitative RT-PCR experiments revealed the hdc and hisRS genes were highly induced under acidic and histidine-rich conditions. This work represents the first description and identification of the hdc-related genes in M. morganii. Results support the hypothesis that the histidine decarboxylation reaction in this prolific histamine producing species may play a role in acid survival. The knowledge of the role and the regulation of genes involved in histidine decarboxylation should improve the design of rational strategies to avoid toxic histamine production in foods. © 2013 Springer Science+Business Media New York., This work was supported by grants AGL2008-01052, AGL2011-22745, and Consolider INGENIO 2010 CSD2007- 00063 FUN-C-FOOD (CICYT), S2009/AGR-1469 (ALIBIRD)(CAM), and RM.2012-00004 (INIA).

© 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)

Immobilized enzymes are preferred over their soluble counterparts due to their robustness in harsh industrial processes; the most stable enzyme derivatives are often produced through multipoint covalent attachment (MCA). However, most enzymes are unable to establish optimal MCA to electrophile-type supports given the heterogeneous distribution and/or low content of primary amino groups on their surfaces; this restricts both the diversity of areas prone to react and the number of attachments to the support. To overcome this we propose combining site-directed immobilization and protein engineering to increase the number of bonds between a specific enzyme surface and a tailor-made support. We applied this novel strategy to engineered mutants of the lipase 2 from Geobacillus thermocatenulatus with one Cys exposed residue, that after genetic amination and/or chemical amination, were immobilized on glyoxyl-disulfide support using a site-directed MCA protocol. Two highly stabilized derivatives of chemically aminated lipase variants, in which site-directed MCA implied the surrounding surface of residues Cys344 or Cys40, were produced: the first one was 2.4-fold more productive than the reference derivative (648 g of hydrolyzed ester); the second derivative was 40% more selective (EPA/DHA molar ratio) and as active (1 μmol g catalyst-1 min-1) as the reference in the production of PUFAs. © 2014 Elsevier Ltd., CICYT (project BIO-2005-6018576), Madrid Region council CAM (grants S0505/PPQ/0344) and CSIC-Spain.