José Miguel Mancheño

Food phenolics and lactic acid bacteria.

Phenolic compounds are important constituents of food products of plant origin. These compounds are directly related to sensory characteristics of foods such as flavour, astringency, and colour. In addition, the presence of phenolic compounds on the diet is beneficial to health due to their chemopreventive activities against carcinogenesis and mutagenesis, mainly due to their antioxidant activities. Lactic acid bacteria (LAB) are autochthonous microbiota of raw vegetables. To get desirable properties on fermented plant-derived food products, LAB has to be adapted to the characteristics of the plant raw materials where phenolic compounds are abundant. Lactobacillus plantarum is the commercial starter most frequently used in the fermentation of food products of plant origin. However, scarce information is still available on the influence of phenolic compounds on the growth and viability of L. plantarum and other LAB species. Moreover, metabolic pathways of biosynthesis or degradation of phenolic compounds in LAB have not been completely described. Results obtained in L. plantarum showed that L. plantarum was able to degrade some food phenolic compounds giving compounds influencing food aroma as well as compounds presenting increased antioxidant activity. Recently, several L. plantarum proteins involved in the metabolism of phenolic compounds have been genetically and biochemically characterized. The aim of this review is to give a complete and updated overview of the current knowledge among LAB and food phenolics interaction, which could facilitate the possible application of selected bacteria or their enzymes in the elaboration of food products with improved characteristics.

Structural analysis of the Laetiporus sulphureus hemolytic pore-forming lectin in complex with sugars.

LSL is a lectin produced by the parasitic mushroom Laetiporus sulphureus, which exhibits hemolytic and hemagglutinating activities. Here, we report the crystal structure of LSL refined to 2.6-Å resolution determined by the single isomorphous replacement method with the anomalous scatter (SIRAS) signal of a platinum derivative. The structure reveals that LSL is hexameric, which was also shown by analytical ultracentrifugation. The monomeric protein (35 kDa) consists of two distinct modules: an N-terminal lectin module and a pore-forming module. The lectin module has a β-trefoil scaffold that bears structural similarities to those present in toxins known to interact with galactose-related carbohydrates such as the hemagglutinin component (HA1) of the progenitor toxin from Clostridium botulinum, abrin, and ricin. On the other hand, the C-terminal pore-forming module (composed of domains 2 and 3) exhibits three-dimensional structural resemblances with domains 3 and 4 of the β-pore-forming toxin aerolysin from the Gram-negative bacterium Aeromonas hydrophila, and domains 2 and 3 from the ϵ-toxin from Clostridium perfringens. This finding reveals the existence of common structural elements within the aerolysin-like family of toxins that could be directly involved in membrane-pore formation. The crystal structures of the complexes of LSL with lactose and N-acetyllactosamine reveal two dissacharide-binding sites per subunit and permits the identification of critical residues involved in sugar binding.

Sticholysins, two pore-forming toxins produced by the Caribbean Sea anemone Stichodactyla helianthus: Their interaction with membranes

Sticholysins (Sts) I and II (StI/II) are pore-forming toxins (PFTs) produced by the Caribbean Sea anemone Stichodactyla helianthus belonging to the actinoporin family, a unique class of eukaryotic PFTs exclusively found in sea anemones. As for the rest of the members of this family, Sts are cysteine-less proteins, with molecular weights around 20 kDa, high isoelectric points (>9.5), and a preference for sphingomyelin-containing membranes. A three-dimensional structure of StII, solved by X-ray crystallography, showed that it is composed of a hydrophobic beta-sandwich core flanked on the opposite sides by two alpha helices comprising residues 14-23 and 128-135. A variety of experimental results indicate that the first thirty N-terminal residues, which include one of the helices, are directly involved in pore formation. This region contains an amphipathic stretch, well conserved in all actinoporins, which is the only portion of the molecule that can change conformation without perturbing the general protein fold; in fact, binding to model membranes only produces a slight increase in the regular secondary structure content of Sts. Sts are produced in soluble form but they readily bind to different cell and model membrane systems such as lipidic monolayers, micelles, and lipid vesicles. Remarkably, both the binding and pore-formation steps are critically dependent on the physico-chemical nature of the membrane. In fact, a large population of toxin irreversibly binds with high affinity in membranes containing sphingomyelin whereas binding in membranes lacking this sphingolipid is relatively low and reversible. The joint presence of SM and cholesterol largely promotes binding and pore formation. Minor amounts of lipids favoring a non-lamellar organization also augment the efficiency of pore formation. The functional pore formed in cellular and model membranes has a diameter of approximately 2.0 nm and is presumably formed by the N-terminal alpha helices of four monomers tilted 31 degrees in relation to the bilayer normal. Experimental evidence supports the hypothesis that sticholysins, as well as equinatoxin II, another actinoporin, form a toroidal pore in membranes in which the polypeptide chains as well as the polar head groups of phospholipids are involved.

Production and physicochemical properties of recombinant Lactobacillus plantarum tannase.

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 degrees C. Addition of Ca(2+) 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.

P-Coumaric Acid Decarboxylase from Lactobacillus Plantarum: Structural Insights Into the Active Site and Decarboxylation Catalytic Mechanism.

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 β‐barrel surrounding an internal cavity. Crystallographic and functional analyses of single‐point mutants of residues located within this cavity have permitted identifying a potential substrate‐binding 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. Proteins 2010.

Characterization of the p-Coumaric Acid Decarboxylase from Lactobacillus plantarum CECT 748T

It was previously reported that cell cultures from Lactobacillus plantarum CECT 748 (T) 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 748 (T). Contrarily to L. plantarum LPCHL2 PDC, the recombinant PDC from L. plantarum CECT 748 (T) is a heat-labile enzyme, showing optimal activity at 22 degrees 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 K(M) 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.

Characterization of a feruloyl esterase from Lactobacillus plantarum.

ABSTRACT 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.

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

A family of restriction enzyme- and ligation-independent cloning vectors has been developed for producing recombinant His-tagged fusion proteins in Escherichia coli. These are based on pURI2 and pURI3 expression vectors which have been previously used for the successful production of recombinant proteins at the milligram scale. The newly designed vectors combines two different promoters (lpp(p)-5 and T7 RNA polymerase Ø10), two different endoprotease recognition sites for the His₆-tag removal (enterokinase and tobacco etch virus), different antibiotic selectable markers (ampicillin and erythromycin resistance), and different placements of the His₆-tag (N- and C-terminus). A single gene can be cloned and further expressed in the eight pURI vectors by using six nucleotide primers, avoiding the restriction enzyme and ligation steps. A unique NotI site was introduced to facilitate the selection of the recombinant plasmid. As a case study, the new vectors have been used to clone the gene coding for the phenolic acid decarboxylase from Lactobacillus plantarum. Interestingly, the obtained results revealed markedly different production levels of the target protein, emphasizing the relevance of the cloning strategy on soluble protein production yield. Efficient purification and tag removal steps showed that the affinity tag and the protease cleavage sites functioned properly. The novel family of pURI vectors designed for parallel cloning is a useful and versatile tool for the production and purification of a protein of interest.

Phenotypic selection and characterization of randomly produced non-haemolytic mutants of the toxic sea anemone protein sticholysin II.

A rapid screening method for haemolytic activity, using blood agar plates, has been developed to analyze randomly produced mutant variants of the pore‐forming protein sticholysin II (Stn II). Those exhibiting a reduced activity were selected and the DNA corresponding to each Stn II variant sequenced. Once the mutation produced was determined, protein variants were isolated and characterized in terms of structure (circular dichroism spectra and thermal stability) and haemolytic activity. Three single mutation protein variants, at residues K19, F106 and Y111, showed a significantly decreased haemolytic activity while their thermostability was identical to that of the wild‐type protein. Considering the obtained data and based on the three‐dimensional structure of the protein, the role of these residues on the mechanism of haemolysis has been analyzed.

Tannin Degradation by a Novel Tannase Enzyme Present in Some Lactobacillus plantarum Strains

ABSTRACT 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.