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Aim To increase our knowledge on the functionality of 6-phospho-β-glucosidases linked to phosphoenolpyruvate-dependent phosphotransferase systems (PTS) that are encountered in high redundancy in the Lactiplantibacillus plantarum WCFS1 genome. Methods and Results Two L. plantarum WCFS1 gene mutants that lacked one of the 6-phospho-β-glucosidases, ∆pbg2 (or ∆lp_0906) or ∆pbg4 (or ∆lp_2777) were constructed and the metabolic impact of these mutations assessed by high-throughput phenotyping (Omnilog). The ∆pbg2 mutant displayed a reduced metabolic performance, having lost the capacity to utilize 20 out of 57 carbon (C)-sources used by the wild-type strain. Conversely, the ∆pbg4 mutant conserved the capacity to metabolize most of the C-sources preferred by the wild type strain. This mutant utilized 56 C-sources albeit the range of substrates used and hence its metabolic profiling differed from that of the WCFS1 strain. The ∆pbg2 mutant notably reduced or abolished the capacity to metabolize substrates related to pentose and glucoronate interconversions and was unable to assimilate fatty acids or nucleosides as sole C-sources for growth. The ∆pbg4 mutant acquired the capacity to utilize efficiently glycogen, indicating an efficient supply of glucose from this source. Conclusion Lactiplantibacillus plantarum gene mutants that lack individual 6-phospho-β-glucosidases display very different carbohydrate utilization signatures showing that these enzymes can be crucial to determine the capacity of L. plantarum to consume different C-sources and hence for the nutrition and physiology of this microorganism.

The conjugation of Au complexes with proteins and enzymes, generating new types of artificial metalloenzymes, has proven to be interesting and effective to obtain materials with improved properties such as higher stability, catalytic activity and selectivity. In this work, a novel method has been developed for the synthesis and design of artificial gold metalloenzymes at 50ºC in aqueous media, using two genetically modified variants of the alkalophilic lipase Geobacillus thermocatenulatus (GTL). The only difference between these two enzymatic variants is the possible coordination of the Au via active site (GTL-114) or Lid site (GTL-193). TEM analysis of the metalloenzymes revealed the formation of Au (0) nanoparticles with different structures (nanowires, nanorods, nanoshells, nanoclusters) and sizes depending on the mutant and the pH used during the synthesis. Characterisation by fluorescence spectroscopy demonstrated that conjugation of the enzyme to Au altered the tertiary structure of the protein. On the other hand, all metalloenzymes showed excellent reductase like activity. Finally, the selectivity of the enzyme-Au bioconjugates was tested in the asymmetric reduction of acetophenone to 1-phenylethanol in aqueous medium at room temperature. The protein environment played a key role in the reactivity and selectivity of the metalloenzymes, obtaining chiral nanoparticles with an enantiomeric excess of up to 39% towards (R)-1-phenylethanol after two hours of reaction using GTL-114 pH 10 as catalyst.

The hydrolysis of plant glucosinolates by myrosinases (thioglucosidases) originates metabolites with chemopreventive properties. In this study, the ability to hydrolyze the glucosinolate sinigrin by cultures or protein extracts of

Lactobacilli are well-studied bacteria that can undergo oxidative selective pressures by plant phenolic compounds (PPCs) in plants, during some food fermentations or in the gastrointestinal tract of animals via dietary inputs. Lactobacilli are known to be more tolerant to PPCs than other bacterial groups and, therefore, must have mechanisms to cope with the effects of these metabolites. In this review, we intend to present what is currently known about the basics beyond the responses of Lactobacillus spp. to individual PPCs. We review the molecular mechanisms that are engaged in the PPC-modulated responses studied to date in these bacteria that have been mainly characterized by system-based strategies, and we discuss their differences and similarities. A wide variety of mechanisms are induced to increase the oxidative stress response highlighting the antimicrobial nature of PPCs. However other uncovered mechanisms that are involved in the response to these compounds are reviewed, including the capacity of PPCs to modulate the expression of molecular functions used by lactobacilli to adapt to host environments. This shows that these phytochemicals can act as more than just antimicrobial agents in the dual interaction with lactobacilli., This work was supported by the Spanish Ministerio de Economía, Industria and Competitividad under Grant numbers AGL2017-84614-C2-2-R and AGL2014-52911 (AEI/FEDER, UE) and CSIC under Grant number ICOOPB20412.

The hydrolysis of plant glucosinolates by myrosinases (thioglucosidases) originates metabolites with chemopreventive properties. In this study, the ability to hydrolyze the glucosinolate sinigrin by cultures or protein extracts of Lactiplantibacillus plantarum WCFS1 was assayed. This strain possesses myrosinase-like activity as sinigrin was partly hydrolyzed by induced cultures but not by protein extracts. The 11 glycoside hydrolase GH1 family proteins, annotated as 6-phospho-β-glucosidases, were the proteins most similar to plant myrosinases. The activity of these proteins was assayed against sinigrin and synthetic glucosides. As expected, none of the proteins assayed possessed myrosinase activity against sinigrin or the synthetic β-thio-glucoside derivative or against the β-glucoside. However, all 11 proteins were active on the phosphorylated-β-glucoside derivative. Moreover, only eight of these proteins were active on phospho-β-thioglucose. These results supported that, in L. plantarum WCFS1, glucosinolates may undergo previous phosphorylation, and GH1 proteins are the glycosidases involved in the hydrolysis of phosphorylated glucosinolates., This work was financially supported by grants AGL2017-84614-C2-1 R and AGL2017-84614-C2-2 R funded by MCIN/AEI/10.13039/501100011033 and by ERDF A way of making Europe. Ana Sánchez-Arroyo is a recipient of the FPI contract PRE2018-083862 funded by MCIN/AEI/10.13039/501100011033 by ESF Investing in your future.

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

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).

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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

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.

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).

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

The production, biochemical characterization, and carbohydrate specificity of LacA β-galactosidase (locus lp_3469) belonging to the glycoside hydrolase family 42 from the probiotic organism Lactobacillus plantarum WCFS1 are addressed. The β-d-galactosidase activity was maximal in the pH range of 4.0-7.0 and at 30-37 °C. High hydrolysis capacity toward the β(1 → 4) linkages between galactose and glucose (lactose) or fructose (lactulose) was found. High efficiency toward galactosyl derivative formation was observed when lactose and glycerol, xylitol, or erythritol were used. Galactosyl derivatives of xylitol were characterized for the first time as 3-O-β-d-galactopyranosyl-xylitol and 1-O-β-d-galactopyranosyl-xylitol, displaying high preference of LacA β-galactosidase for the transfer of galactosyl residues from lactose to the C1 or C3 hydroxyl group of xylitol. These results indicate the feasibility of using LacA β-galactosidase for the synthesis of different galactosyl-polyols, which could be promising candidates for beneficial and appealing functional and technological applications such as novel prebiotics or hypocaloric sweeteners., This work has been financed by the Spanish Ministry of Economy, Industry and Competitiveness (Projects AGL2017- 84614-C2-1-R and AGL2017-84614-C2-2-R).

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 article belongs to the Special Issue Catalysts for a Green Chemistry: A Themed Issue Honoring Professor Roger Sheldon for His Contribution in the Field of the Green Chemistry with the Introduction of the E Factor., Different materials containing carboxylic groups have been functionalized with geranyl-amine molecules by using an EDC/NHS strategy. Chemical modification of the support was confirmed by XRD, UV-spectrophotometer, and FT-IR. This geranyl-functionalized material was successfully applied for four different strategies of site-selective immobilization of proteins at room temperature and aqueous media. A reversible hydrophobic immobilization of proteins (lipases, phosphoglucosidases, or tyrosinase) was performed in neutral pH in yields from 40 to >99%. An increase of the activity in the case of lipases was observed from a range of 2 to 4 times with respect to the initial activity in solution. When chemically or genetically functionalized cysteine enzymes were used, the covalent immobilization, via a selective thiol-alkene reaction, was observed in the presence of geranyl support at pH 8 in lipases in the presence of detergent (to avoid the previous hydrophobic interactions). Covalent attachment was confirmed with no release of protein after immobilization by incubation with hydrophobic molecules. In the case of a selenium-containing enzyme produced by the selenomethionine pathway, the selective immobilization was successfully yielded at acidic pH (pH 5) (89%) much better than at pH 8. In addition, when an azido-enzyme was produced by the azide–homoalanine pathway, the selective immobilization was successful at pH 6 and in the presence of CuI for the click chemistry reaction., This work was sponsored by the Spanish Government (Project No. AGL2017-84614-C2-2-R, AEI/FEDER) and CSIC Project No. CSIC-PIE 201880E011.

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.

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.

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).