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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).

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.

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.

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.

[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

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.

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.

Endogenous galactitol-1-phosphate 5-dehydrogenase (GPDH) (EC 1.1.1.251) from Escherichia coli spontaneously interacts with Ni 2+-NTA matrices becoming a potential contaminant for recombinant, target His-tagged proteins. Purified recombinant, untagged GPDH (rGPDH) converted galactitol into tagatose, and d-tagatose-6-phosphate into galactitol-1-phosphate, in a Zn 2+- and NAD(H)-dependent manner and readily crystallized what has permitted to solve its crystal structure. In contrast, N-terminally His-tagged GPDH was marginally stable and readily aggregated. The structure of rGPDH revealed metal-binding sites characteristic from the medium-chain dehydrogenase/reductase protein superfamily which may explain its ability to interact with immobilized metals. The structure also provides clues on the harmful effects of the N-terminal His-tag. Structured summary of protein interactions: GPDH and GPDH bind by molecular sieving (View interaction) GPDH and GPDH bind by x-ray crystallography (View interaction) GPDH and GPDH bind by cosedimentation in solution (View interaction). © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved., JMM and RM thank the Ministerio de Ciencia e Innovación for their research grantsBFU2010-17929, CSD2006-00015 and AGL2011-22745, CSD2007-00063 FUN-C-FOOD and Comunidad de Madrid S2009/AGR-1469, respectively.

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.

A new strategy has been developed for site-directed immobilization/rigidification of genetically modified enzymes through multipoint covalent attachment on bifunctional disulfide-glyoxyl supports. Here the mechanism is described as a two-step immobilization/rigidification protocol where the enzyme is directly immobilized by thiol-disulfide exchange between the β-thiol of the single genetically introduced cysteine and the few disulfide groups presented on the support surface (3 μmol/g). Afterward, the enzyme is uniquely rigidified by multipoint covalent attachment (MCA) between the lysine residues in the vicinity of the introduced cysteine and the many glyoxyl groups (220 μmol/g) on the support surface. Both site-directed immobilization and rigidification have been possible only on these novel bifunctional supports. In fact, this technology has made possible to elucidate the protein regions where rigidification by MCA promoted higher protein stabilizations. Hence, rigidification of vicinity of position 333 from lipase 2 from Geobacillus thermocatenulatus (BTL2) promoted a stabilization factor of 33 regarding the unipunctual site-directed immobilized derivative. In the same context, rigidification of penicillin G acylase from E. coli (PGA) through position β201 resulted in a stabilization factor of 1069. Remarkably, when PGA was site-directed rigidified through that position, it presented a half-life time of 140 h under 60% (v/v) of dioxane and 4 °C, meaning a derivative eight times more stable than the PGA randomly immobilized on glyoxyl-disulfide agarose. Herein we have opened a new scenario to optimize the stabilization of proteins via multipoint covalent immobilization, which may represent a breakthrough in tailor-made tridimensional rigidification of proteins.

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.

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.

[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.

© 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.

During oral passage, food tannins interact with the microbiota present in the oral cavity. Atopobium parvulum strains are inhabitants of the human oral cavity. A gene encoding a protein similar to bacterial tannases is present in A. parvulum strains. The tanAAp (apar_1020) gene was cloned and expressed in Escherichia coli BL21 (DE3). The overproduced TanAAp protein was purified to homogeneity. It exhibited optimal activity at pH6.0 and broad temperature range, being these properties compatible with its action during food oral processing. However, purified TanAAp protein presented the lowest specific activity among bacterial tannases (3.5U/mg) and was unable to hydrolyze complex tannin, such as tannic acid. These biochemical properties discard a main role of TanAAp in the breakdown of complex food tannins during oral processing. © 2014 Elsevier Ltd., This work was financially supported by grants AGL2011-22745, S2009/AGR-1469 (ALIBIRD) (Comunidad de Madrid), and RM2012-00004 (INIA).

The ability of an inulosucrase (IS) from Lactobacillus gasseri DSM 20604 to synthesize fructooligosaccharides (FOS) and maltosylfructosides (MFOS) in the presence of sucroseand sucrose-maltose mixtures was investigated after optimization of synthesis conditions,including enzyme concentration, temperature, pH, and reaction time. The maximum formationof FOS, which consist of β-2,1-linked fructose to sucrose, was 45% (in weight with respect to the initial amount of sucrose) and was obtained after 24 h of reaction at 55°C in the presence of sucrose (300 g liter-1) and 1.6 U ml-1 of IS-25mM sodium acetate buffer-1 mM CaCl2 (pH 5.2). The production of MFOS was also studied as a function of the initial ratios of sucrose to maltose (10:50, 20:40, 30:30, and 40:20, expressed in g 100 ml-1). The highest yield in total MFOS was attained after 24 to 32 h of reaction time and ranged from 13% (10:50 sucrose/maltose) to 52% (30:30 sucrose/maltose) in weight with respect to the initial amount of maltose. Nuclear magnetic resonance (NMR) structural characterization indicated that IS from L. gasseri specifically transferred fructose moieties of sucrose to either C-1 of the reducing end or C-6 of the nonreducing end of maltose. Thus, the trisaccharide erlose [α-D-glucopyranosyl-(1→4)-α-D-glucopyranosyl-(1→2)-β-D-fructofuranoside] was the main synthesized MFOS followed by neo-erlose [β-D-fructofuranosyl-(2→6)-α-D-glucopyranosyl-(1→4)-α-D-glucopyranose]. The formation of MFOS with a higher degree of polymerization was also demonstrated by the transfer of additional fructose residues to C-1 of either the β-2,1-linked fructose or the β-2,6-linked fructose to maltose, revealing the capacity of MFOS to serve as acceptors. © 2013, American Society for Microbiology., This work has been financed by projects AGL2011-27884 and Consolider Ingenio 2010 FUN-C-FOOD CSD2007-00063 (both from Ministerio de Ciencia e Innovación) and project POII10-0178-4685 from Junta de Comunidades de Castilla-La Mancha and the European Regional Development Development Fund [ERDF]). M.D.-M. is supported by CSIC through the JAE-Pre Programme cofunded by the European Social Fund (ESF). M.H. thanks MICINN for his “Ramón y Cajal” contract.

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

In bio-redox cascade reactions that are immobilized on porous supports, mass-transfer limitations may impede the effective concentration of the cofactor around the corresponding dehydrogenases. This main drawback has been addressed by the co-immobilization of both the main and recycling dehydrogenases. Herein, we report tailor-made co-immobilization procedures to assemble three different bio-redox orthogonal cascades invitro (two selective reductions and one selective oxidation) with insitu cofactor-regeneration. However, the co-immobilization itself does not guarantee the success of the biotransformation because the same co-immobilization chemistry may not be suitable for the two enzymes that are involved in the bio-redox cascade. Therefore, our co-immobilization system was optimized for each bi-enzymatic cascade. In all cases, the optimized co-immobilization procedure was more efficient in the biocatalytic cascade than if the two dehydrogenases were immobilized on two different carriers. In one specific case (one thermophilic cascade), the co-immobilization of an optimal ratio of main/recycling dehydrogenases (1:5) on the same carrier resulted in a biocatalyst that was able to recycle NADH up to 9000times per equivalent of substrate in 1hour at 55°C. Moreover, uniform distributions of both dehydrogenases across the porous surface also enhanced the recycling efficiency of the cofactor 1.5-fold versus cascades in which the enzymes were not uniformly distributed across the same porous surface, presumably because of vicinal cooperation effects. Hence, this system for the co-immobilization of bi-enzymatic systems may be extended to other biocatalytic cascades, thereby opening a window for the optimization of other multi-enzyme biotransformations in which cofactor-recycling is necessary. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim., Financial support from the Madrid Region council CAM (grant no. S0505/PPQ/0344) and the Spanish Ministry of Science (Juan de la Cierva fellowship and grant BIO-2008–01481)

An efficient chemoselective method for the creation of semisynthetic lipases by site-specific incorporation of tailor-made peptides on the lipase-lid site was developed. These new enzymes showed excellent improved specificity and regio- or enantioselectivity in different biotransformations. © 2012 The Royal Society of Chemistry., Spanish National Research Council (CSIC) and by grants BFU2011-25326, BFU2011-24595

Tyramine poisoning is caused by the ingestion of food containing high levels of tyramine, a biogenic amine. Any foods containing free tyrosine are subject to tyramine formation if poor sanitation and low quality foods are used or if the food is subject to temperature abuse or extended storage time. Tyramine is generated by decarboxylation of the tyrosine through tyrosine decarboxylase (TDC) enzymes derived from the bacteria present in the food. Bacterial TDC have been only unequivocally identified and characterized in Gram-positive bacteria, especially in lactic acid bacteria. Pyridoxal phosphate (PLP)-dependent TDC encoding genes (tyrDC) appeared flanked by a similar genetic organization in several species of lactic acid bacteria, suggesting a common origin by a single mobile genetic element. Bacterial TDC are also able to decarboxylate phenylalanine to produce phenylethylamine (PEA), another biogenic amine. The molecular knowledge of the genes involved in tyramine production has led to the development of molecular methods for the detection of bacteria able to produce tyramine and PEA. These rapid and simple methods could be used for the analysis of the ability to form tyramine by bacteria in order to evaluate the potential risk of tyramine biosynthesis in food products. © 2012 Copyright Taylor and Francis Group, LLC., This work was supported by grants AGL2008-01052, AGL2011-22745, and CONSOLIDER INGENIO 2010 CSD2007-00063 FUN-C-FOOD (CICYT), RM2008-00002 (INIA), and S2009/AGR-1469 (ALIBIRD) (CAM). We are grateful to M. V. Santamaría. J. M. Landete was a recipient of a postdoctoral fellowship from the MEC.

In this work, biogenic amine production (histamine, tyramine and putrescine) by a collection of 74 lactic acid bacteria of aquatic origin has been investigated by means of amino acid decarboxylation by growth on decarboxylase differential medium, biogenic amine detection by thin-layer chromatography (TLC) and decarboxylase gene detection by PCR. None of the evaluated strains showed neither production of histamine and putrescine, nor presence of the genetic determinants encoding the corresponding decarboxylase activities. However, the tyrosine decarboxylase gene (tdc) was present in all the enterococcal strains, and tyramine production was detected by TLC in all of them but Enterococcus faecium BCS59 and MV5. Analysis of the tyrosine decarboxylase operon of these strains revealed the presence of an insertion sequence upstream tdc that could be responsible for their lack of tyrosine decarboxylase activity. © 2011 Elsevier B.V., This work was partially supported by projects AGL2009-08348ALI and Consolider INGENIO 2010 CSD2007-00063 FUN-C-FOOD from the Ministerio de Ciencia y Tecnología (MCYT), Spain; Spanish–Portuguese Integrated Action HP2008-0070 from the Ministerio de Ciencia e Innovación (MICINN), Spain; S2009/AGR-1469 and S2009/AGR-1489 from the Comunidad de Madrid (CAM), Spain; and RM2008-00002 from the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Spain. G. Landeta is a recipient of a predoctoral fellowship from the Consejo Nacional de Ciencia y Tecnología (CONACyT), Mexico. P. Natale is acknowledged for artwork.

The influence of additives on the activity of different covalently and site-specific chemically modified immobilized preparations of a lipase from Geobacillus thermocatenulatus (BTL2) was investigated with a view to obtain a very high active biocatalyst. Non-ionic surfactant and co-solvents at different concentration range were applied. The CNBr-BTL2 immobilized preparation, a very mild immobilized enzyme with similar properties to the soluble enzyme, exhibited an increase in activity of 3 fold in the presence of 20% (v/v) co-solvent (e.g., 1,4-dioxane) and 2.6 fold when Triton X-100 (v/v) was added in the hydrolysis of p-nitrophenylbutyrate. This immobilized preparation was hyper-activated in the presence of both additives although without a synergistic effect. The CNBr-BTL2 modified with polymers showed mild hyperactivation in the presence of each additives and even a synergy in the presence of both. In the best of cases, the HOOC-PEG1500-CNBr-BTL2 preparation showed up to 11 fold higher activity in the presence of additives combination than in absence of them. The glyoxyl-BTL2 preparation was hyper-activated in a similar way than CNBr-BTL2 in the presence of detergents but much less with co-solvents. However, the modified glyoxyl-BTL2 preparations were hyper-activated with solvent (2 fold) but not with detergent. An increase of 3 fold in activity for the modified glyoxyl-BTL2 preparations was observed in the presence of both additives. © 2011 Elsevier B.V. All rights reserved.

Biogenic amines are low molecular weight organic bases frequently found in wine. Several toxicological problems resulting from the ingestion of wine containing biogenic amines have been described. In wine, histamine, tyramine, and putrescine are mainly produced by the decarboxylation of the amino acid histidine, tyrosine, and ornithine, respectively, by lactic acid bacteria action. The bacterial ability to decarboxylate amino acids is highly variable, and therefore the detection of bacteria possessing amino acid decarboxylase activity is important to prevent biogenic amine accumulation in wine. Molecular methods for the early and rapid detection of these producer bacteria are becoming an alternative to traditional culture methods. Moreover, quantitative PCR methods are useful to enumerate biogenic amine-producer bacteria on wine. Molecular methods detect potential biogenic amine risk formation in wine before the amine is produced. This review will cover the molecular methods proposed in the literature for the detection of biogenic amine-producing bacteria in wine. These methods could improve winemaking control in order to avoid biogenic amine production, This work was supported by grants RM2008- 00002 (Instituto Nacional de Investigación Agraria y Alimentaría), AGL2008-01052, Consolider INGENIO 2010 CSD2007-00063 FUNC- FOOD (Comisión Interministerial de Ciencia y Tecnología), and S-0505/AGR/000153 and S2009/AGR-1469 (ALIBIRD) (Comunidad de Madrid).

Phenolic acid decarboxylase (PAD) catalyzes the synthesis of vinyl phenols from hydroxycinnamic acids. The gene encoding PAD from Lactobacillus brevis was cloned and expressed as a fusion protein in Escherichia coli. The recombinant PAD enzyme is a heat-labile enzyme that functions optimally at 22 C and pH 6.0. The purified enzyme did not show thermostability at temperatures above 22 C. L. brevis PAD is able to decarboxylate exclusively the hydroxycinnamic acids, such as p-coumaric, caffeic, and ferulic acids, with Km values of 0.98, 0.96, and 0.78 mM, respectively. The substrate specificity exhibited by L. brevis PAD is similar to the PAD isolated from Bacillus subtilis and B. pumilus, but different from that of L. plantarum and Pediococcus pentosaceus. As the C-terminal region may be involved in determining PAD substrate specificity and catalytic capacity, amino acid differences among these proteins could explain the differences observed. The substrate specificity shown by L. brevis PAD shows promise for the synthesis of high-added value products from plant wastes, This work was supported by grants AGL2005- 00470, AGL2008-01052, Consolider INGENIO 2010 CSD2007- 00063 FUN-C-FOOD (Comisión Interministerial de Ciencia y Tecnología ), RM2008-00002 (Instituto Nacional de Investigación Agraria y Alimentaría), and S-0505/AGR/000153 and S2009/AGR-1469 (ALIBIRD) (Comunidad de Madrid). We are grateful to M. V. Santamaría and J. M. Barcenilla. 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 Ministerio de Ciencia e Innovación.

p-Coumaric acid decarboxylases (PDCs) catalyze the nonoxidative decarboxylation of hydroxycinnamic acids to generate the corresponding vinyl derivatives. Despite the biotechnological relevance of PDCs in food industry, their catalytic mechanism remains largely unknown. Here, we report insights into the structural basis of catalysis for the homodimeric PDC from Lactobacillus plantarum (LpPDC). The global fold of LpPDC is based on a flattened b-barrel surrounding an internal cavity. Crystallographic and functional analyses of single-point mutants of residues located within this cavity have permitted identifying a potential substratebinding pocket and also to provide structural evidences for rearrangements of surface loops so that they can modulate the accessibility to the active site. Finally, combination of the structural and functional data with in silico results enables us to propose a two-step catalytic mechanism for decarboxylation of p-coumaric acid by PDCs where Glu71 is involved in proton transfer, and Tyr18 and Tyr20 are involved in the proper substrate orientation and in the release of the CO2 product, The authors acknowledge access to ESRF beamlines ID23-1, ID29, and BM16, and local support by A.M. Gonc¸alves (ID23-1), X. Thibault (ID29), and G.C. Fox (BM16). H. Rodrı´guez is a recipient of an I3P predoctoral fellowship from the CSIC

Tannase is an enzyme with important biotechnological applications in the food industry. Previous studies have identified the tannase encoding gene in Lactobacillus plantarum and also have reported the description of the purification of recombinant L. plantarum tannase through a protocol involving several chromatographic steps. Here, we describe the high-yield production of pure recombinant tannase (17 mg/L) by a one-step affinity procedure. The purified recombinant tannase exhibits optimal activity at pH 7 and 40 C. Addition of Ca2þ to the reaction mixture greatly increased tannase activity. The enzymatic activity of tannase was assayed against 18 simple phenolic acid esters. Only esters derived from gallic acid and protocatechuic acid were hydrolyzed. In addition, tannase activity was also assayed against the tannins tannic acid, gallocatechin gallate, and epigallocatechin gallate. Despite L. plantarum tannase representing a novel family of tannases, which shows no significant similarity to tannases from fungal sources, both families of enzymes shared similar substrate specificity range. The physicochemical characteristics exhibited by L. plantarum recombinant tannase make it an adequate alternative to the currently used fungal tannases, This work was supported by grants AGL2005-00470, AGL2008-01052, BFU2007-67404/BMC, Consolider INGENIO 2010 CSD2007-00063 FUN-C-FOOD, and Consolider INGENIO 2007 Factoria de Crystalización (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.

8 pages.-- PMID: 19056729 [PubMed].-- Available online Dec 3, 2008., Supporting information available at: http://www.jbc.org/cgi/content/full/M808268200/DC1, The bacterial thermoalkalophilic lipases that hydrolyze saturated fatty acids at 60–75°C and pH 8–10 are grouped as the lipase family I.5. We report here the crystal structure of the lipase from Geobacillus thermocatenulatus, the first structure of a member of the lipase family I.5 showing an open configuration. Unexpectedly, enzyme activation involves large structural rearrangements of around 70 amino acids and the concerted movement of two lids, the α6- and α7-helices, unmasking the active site. Central in the restructuring process of the lids are both the transfer of bulky hydrophobic residues out of the N-terminal end of the α6-helix and the incorporation of short side chain residues to the α6 C-terminal end. All these structural changes are stabilized by the Zn(2+)-binding domain, which is characteristic of this family of lipases. Two detergent molecules are placed in the active site, mimicking chains of the triglyceride substrate, demonstrating the position of the oxyanion hole and the three pockets that accommodate the sn-1, sn-2, and sn-3 fatty acids chains. The combination of structural and biochemical studies indicate that the lid opening is not mediated by temperature but triggered by interaction with lipid substrate., This work was supported by Grant BFU2005-01645 from Dirección General de Investigación and by "Factoria de Cristalización," CONSOLIDER INGENIO-2010.

In this paper, the stabilization of a lipase from Bacillus thermocatenulatus (BTL2) by a new strategy is described. First, the lipase is selectively adsorbed on hydrophobic supports. Second, the carboxylic residues of the enzyme are modified with ethylenediamine, generating a new enzyme having 4-fold more amino groups than the native enzyme. The chemical amination did not present a significant effect on the enzyme activity and only reduced the enzyme half-life by a 3-4-fold factor in inactivations promoted by heat or organic solvents. Next, the aminated and purified enzyme is desorbed from the support using 0.2% Triton X-100. Then, the aminated enzyme was immobilized on glyoxyl-agarose by multipoint covalent attachment. The immobilized enzyme retained 65% of the starting activity. Because of the lower p K of the new amino groups in the enzyme surface, the immobilization could be performed at pH 9 (while the native enzyme was only immobilized at pH over 10). In fact, the immobilization rate was higher at this pH value for the aminated enzyme than that of the native enzyme at pH 10. The optimal stabilization protocol was the immobilization of aminated BTL2 at pH 9 and the further incubation for 24 h at 25 degrees C and pH 10. This preparation was 5-fold more stable than the optimal BTL2 immobilized on glyoxyl agarose and around 1200-fold more stable than the enzyme immobilized on CNBr and further aminated. The catalytic properties of BTL2 could be greatly modulated by the immobilization protocol. For example, from (R/S)-2- O-butyryl-2-phenylacetic acid, one preparation of BTL2 could be used to produce the S-isomer, while other preparation produced the R-isomer.

Resumen del póster presentado al 2nd Annual Meeting COST 928, celebrado en Istanbul (Turquía) del 15 al 17 de octubre de 2008., In foods, tannins are considered nutritionally undesirable. However, the intake of a small quantity of the right kind oftannins may be beneficial to human health. It has been found that the higher the molar mass of tannin molecules, the stronger the antinutritional effects and the lower the biological activities. Microbial degradation is one of the efficient ways to degrade large molecular tannins into smaller molecular tannins with valuable bioactivities. In this work we evaluated the ability of Lactobacillus plantarum to degrade hydrolysable tannins. Significative reduction of tannic acid was not observed during incubation in the presence of L. plantarum cells after 7 days incubation. However, tannic acid was effectively degraded by cell-free extracts containing soluble proteins from L. plantarum. By HPLC analysis, almost a complete tannic acid degradation was observed in the three commercial tannic acid samples assayed. By using HPLC-DADIESI-MS, we partially determined the composition of tannic acid from Quercus infectoria galls. This tannic acid is a gallotannin mainly composed of monomers to tetramers of gallic acid. We studied the mechanism of its degradation by L. plantarum. The results indicated that L. plantarum degrades gallotannins by depolymerization of high molecular weight tannins and a reduction of low molecular weight tannins. Gallic acid and pyrogallol were detected as final metabolic intermediates.

Bacillus thermocatenulatus lipase 2 (BTL2) is a thermoalkalophilic lipase that has been reported as an enantioselective biocatalyst for diverse reactions and that heads a group of enzymes that share high resistance towards many inactivation agents (heat, organic solvents, pH etc.). This makes BTL2 an important research target because of its potential industrial applications. BTL2 was cloned and overexpressed in Escherichia coli, purified and concentrated for crystallization using the sitting-drop vapour-diffusion method at 291 K. Crystals grew from a mixture of 13% MPD and 0.2 M ammonium acetate in 0.05 M sodium citrate pH 5.5-5.6. The crystals, which belonged to the orthorhombic space group I222 with unit-cell parameters a = 73.07, b = 129.08, c = 127.49 Å, allowed the collection of an X-ray data set to 2.2 Å resolution.

In foods, tannins are considered nutritionally undesirable. Spectrophotometric methods have been used to detect tannin degradation by L. plantarum strains isolated from food substrates. Enzymatic degradation of tannic acid by L. plantarum CECT 748T was examined in liquid cultures and in cell-free extracts by HPLC. Significative reduction of tannic acid was not observed during incubation in the presence of L. plantarum cells after 7 days incubation. However, tannic acid was effectively degraded by cell-free extracts of L. plantarum during 16 h incubation. We have partially characterized L. plantarum tannase activity by measuring its esterase activity on methyl gallate. Tannase activity was optimal at pH 5.0 and 30 °C, and showed nearly 75% of the maximal activity at 50 °C. The biochemical characteristics showed by L. plantarum tannase are considered favourable for tannin biodegradation in the food-processing industry., This work was supported by grants AGL2005-00470 (CICYT), FUN-C-FOOD Consolider 25506 (MEC), RM03-002 (INIA), and S-0505/AGR/000153 (CAM). H. Rodríguez is a recipient of a I3P predoctoral fellowship from the CSIC.

The potential to produce biogenic amines was investigated for 56 coagulase-negative staphylococci isolated during industrial Spanish dry-cured ham processes. The presence of biogenic amines from bacterial cultures was determined by thin-layer chromatography. The percentage of strains that decarboxylated amino acids was very low (3.6%). The only staphylococci with aminogenic capacity were an histamine-producing Staphylococcus capitis strain, and a Staphylococcus lugdunensis strain that simultaneously produced putrescine and cadaverine. In both strains, PCR was used to confirm the presence of the genes encoding the amino acid decarboxylases responsible for the synthesis of these amines. This study reveals that production of biogenic amines is not a widely distributed property among the staphylococci isolated from Spanish dry-cured hams, This work was supported by Grants AGL2005-00470 (CICYT), RM03-002 (INIA), and ALIBIRD-CAM S-0505/AGR/000153 (CAM). The technical assistance of M.V. Santamaría is greatly appreciated. G. Landeta is a recipient of a predoctoral fellowship from CONACyT., S0505/AGR-000153/ALIBIRD

Biogenic amines are low molecular weight organic bases that can be detected in raw and processed foods. Several toxicological problems resulting from the ingestion of food containing biogenic amines have been described. Biogenic amines are mainly produced by the decarboxylation of certain amino acids by microbial action. Since the ability of microorganisms to decarboxylate amino acid is highly variable, being in most cases strain-specific, the detection of bacteria possessing amino acid decarboxylase activity is important to estimate the risk of biogenic amine food content and to prevent biogenic amine accumulation in food products. Molecular methods for the early and rapid detection of these producer bacteria are becoming an alternative to traditional culture methods. PCR methods offer the advantages of speed, sensitivity, simplicity and specific detection of amino acid decarboxylase genes. Moreover, these molecular methods detect potential biogenic amine risk formation in food before the amine is produced. The aim of the present review is to give a complete overview of the molecular methods proposed in the literature for the detection of biogenic amine-producing bacteria. These genetic procedures allow the introduction of early control measures to avoid the development of these bacteria., This work was supported by grant RM03-002 (INIA), AGL2005-00470 (CICYT), and S-0505/AGR/000153 (CAM).

This work deals with the development of a new bioprocess for the efficient synthesis of lactosucrose, a potential prebiotic oligosaccharide with a high value-added, from two important and inexpensive agro-industrial by-products such as tofu whey and cheese whey permeate. The bioconversion is driven by the ability of the enzyme levansucrase SacB from Bacillus subtilis CECT 39 to transfructosylate lactose contained in the cheese whey permeate by using not only sucrose but also raffinose and stachyose, which are present in considerable amounts in the tofu whey, as suitable donors of fructosyl moieties. The maximum lactosucrose concentration obtained from both by-products was 80.1 g L-1 after a short reaction time 120 min at 37°C, leading to productivity and specific productivity values of 40.1 g lactosucrose L-1 h-1 and 80.1 mg lactosucrose U enzyme−1 h−1, respectively. Findings contained in this work could provide a new strategy to valorize agro-industrial by-products as cheese whey permeate and, specially, tofu whey by means of their use as renewable resources in the enzymatic synthesis of bioactive oligosaccharides., This work has been financed by project AGL2011-27884 from Ministerio de Economía y Competitividad.

The genome of the lactic acid bacterium Lactobacillus plantarum WCFS1 reveals the presence of a rich repertoire of esterases and lipases highlighting their important role in cellular metabolism. Among them is the carboxylesterase LpEst1 a bacterial enzyme related to the mammalian hormone-sensitive lipase, which is known to play a central role in energy homeostasis. In this study, the crystal structure of LpEst1 has been determined at 2.05 Å resolution; it exhibits an αβ-hydrolase fold, consisting of a central β-sheet surrounded by α-helices, endowed with novel topological features. The structure reveals a dimeric assembly not comparable with any other enzyme from the bacterial hormone-sensitive lipase family, probably echoing the specific structural features of the participating subunits. Biophysical studies including analytical gel filtration and ultracentrifugation support the dimeric nature of LpEst1. Structural and mutational analyses of the substrate-binding pocket and active site together with biochemical studies provided insights for understanding the substrate profile of LpEst1 and suggested for the first time the conserved Asp173, which is adjacent to the nucleophile, as a key element in the stabilization of the loop where the oxyanion hole resides., We thank the ESRF (Grenoble, France) for provision of synchrotron radiation facilities (ID14-4 and ID29 beamlines, respectively). Y.A.,M.E-T., I.A. and A.C-C are recipients of the following fellowships: CSIC-CITMA, JAE Predoc (CSIC), FPU (MEC) and FPU (MEC), respectively.

A search to identify proteins with high affinity for choline-containing pneumococcal cell walls (choline-binding proteins) has permitted the localization, cloning, sequencing, and overexpression of a gene (pce), coding for a protein (Pce) that liberates phosphorylcholine from purified cell walls of Streptococcus pneumoniae. The pce gene of the pneumococcal strain R6 encodes a protein of 627 amino acids with a predicted Mr of 72,104. Pce can remove a maximum of 20% phosphorylcholine residues from the cell wall teichoic acid. In silico analysis of Pce shows a modular organization of the enzyme where the choline-binding domain, involved in cell wall substrate recognition, and the catalytic domain are located at the carboxy- and amino-terminal moieties of the protein, respectively. Remarkably, a long tail of 85 amino acids follows the carboxy-terminal domain, a structural feature that had not been described for the published choline-binding proteins. The carboxy-terminal moiety of Pce is assembled by 10 repeated motifs, and the protein has also a cleavable signal peptide of 25 amino acids that renders after its cleavage a mature protein of 69,426 Da (602 amino acids). The pce gene has been expressed in Escherichia coli, and Pce was active when assayed on pneumococcal walls. We have also found that the signal peptide of Pce was functional in E. coli. Biochemical analysis suggested that Pce is the teichoic acid phosphorylcholine esterase of S. pneumoniae that had been biochemically characterized previously. Construction of two pce pneumococcal mutants (R6D and M31D) by insertion-duplication mutagenesis revealed that these mutants grew at a doubling-time similar to those of the parental strains of the wild-type R6 and the lytA-mutant M31, respectively. R6D and M31D were morphologically indistinguishable from the parental strains when whole-mounted cells were observed under the electron microscope and exhibited levels of competence for genetic transformation slightly lower than those reported for R6 and M31.