bLactobacillus 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. Phenolic acids are abundant, naturally occurring molecules that contribute to the rigidity of plant cell walls. Hydroxycinnamic acids, such as ferulic, sinapic, caffeic, and p-coumaric acids, are found both covalently attached to the cell wall and as soluble forms in the cytoplasm. Esters and amides are the most frequently reported types of conjugates, whereas glycosides occur only rarely (1). Hydroxycinnamates are found in numerous plant foods and in significant quantities in agroindustry-derived by-products. The industrial use of hydroxycinnamates has attracted growing interest since they and their conjugates were shown to be bioactive molecules possessing potential antioxidant activities and health benefits. The removal of these phenolic compounds and the breakdown of the ester linkages between polymers allow their exploitation for numerous industrial and food applications.Feruloyl esterases, also known as ferulic acid esterases, cinnamic acid esterases, or cinnamoyl esterases, are the enzymes involved in the release of phenolic compounds, such as ferulic, pcoumaric, caffeic, and sinapic acids, from plant cell walls (2). In human and ruminal digestion, feruloyl esterases are important to de-esterify dietary fiber, releasing hydroxycinnamates and derivatives, which have been shown to have positive effects, such as antioxidant, anti-inflammatory, and antimicrobial activities (3). They are also involved in colonic fermentation, where their activities in the microbiota improve the breakdown of ester bonds in hydroxycinnamates (3). The biological properties of hydroxycinnamates depend on their absorption and metabolism. Although there is evidence that food hydroxycinnamates are degraded by the gut microbiota, only limited information on the microorganisms and enzymes involved in this degradation is currently available.Feruloyl esterases able to hydrolyze hydroxycinnamates have been found in lactic acid bacteria isolated from foods and from the human intestinal microbiota, such as some str...

bLactobacillus 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 (TanB Lp , formerly called TanLp1) was previously identified and biochemically characterized. In this study, we report the identification and characterization of a novel tannase (TanA Lp ). While all 29 L. plantarum strains analyzed in the study possess the tanB Lp gene, the gene tanA Lp was present in only four strains. Upon methyl gallate exposure, the expression of tanB Lp was induced, whereas tanA Lp expression was not affected. TanA Lp showed only 27% sequence identity to TanB Lp , but the residues involved in tannase activity are conserved. Optimum activity for TanA Lp was observed at 30°C and pH 6 in the presence of Ca 2؉ ions. TanA Lp was able to hydrolyze gallate and protocatechuate esters with a short aliphatic alcohol substituent. Moreover, TanA Lp was able to fully hydrolyze complex gallotannins, such as tannic acid. The presence of the extracellular TanA Lp tannase in some L. plantarum strains provides them an advantage for the initial degradation of complex tannins present in plant environments.

bLactobacillus plantarum is the lactic acid bacterial species most frequently found in the fermentation of food products of plant origin on which phenolic compounds are abundant. L. plantarum strains showed great flexibility in their ability to adapt to different environments and growth substrates. Of 28 L. plantarum strains analyzed, only cultures from 7 strains were able to hydrolyze hydroxycinnamic esters, such as methyl ferulate or methyl caffeate. As revealed by PCR, only these seven strains possessed the est_1092 gene. When the est_1092 gene was introduced into L. plantarum WCFS1 or L. lactis MG1363, their cultures acquired the ability to degrade hydroxycinnamic esters. These results support the suggestion that Est_1092 is the enzyme responsible for the degradation of hydroxycinnamic esters on the L. plantarum strains analyzed. The Est_1092 protein was recombinantly produced and biochemically characterized. Surprisingly, Est_1092 was able to hydrolyze not only hydroxycinnamic esters, since all the phenolic esters assayed were hydrolyzed. Quantitative PCR experiments revealed that the expression of est_1092 was induced in the presence of methyl ferulate, an hydroxycinnamic ester, but was inhibited on methyl gallate, an hydroxybenzoic ester. As Est_1092 is an enzyme active on a broad range of phenolic esters, simultaneously possessing feruloyl esterase and tannase activities, its presence on some L. plantarum strains provides them with additional advantages to survive and grow on plant environments. Lactobacillus plantarum is a highly versatile lactic acid bacterial species found in many different ecological niches, such as vegetables, meat, fish, and dairy products, as well as in the gastrointestinal tract (1). The genome of L. plantarum strain WCFS1 was the first to be fully sequenced, and it was, in fact, the first of any of the Lactobacillus genomes to be published (2). When the genome diversity of L. plantarum on a full genome scale was analyzed, it was revealed that L. plantarum strains are predicted to lack 9 to 20% of the genes of the L. plantarum WCFS1 reference genome, and about 50 genes appeared to be specific to strain WCFS1, as they were not found in any other strain (1). This variability confirms the flexibility of L. plantarum in its ability to adapt to different environments and growth substrates.Phenolic compounds are important constituents of food products of plant origin, as they are related to the sensory characteristics of the food and are beneficial to consumer health (3). Therefore, it is interesting to know the metabolic pathways of biosynthesis or degradation of these compounds in bacteria. L. plantarum is the lactic acid bacterium that is the most frequently found in the fermentation of food products of plant origin, being the bacterial model for the study of phenolic compound metabolism (4). Among these compounds, the metabolism of phenolic esters is greatly relevant, as they are widely spread throughout the plant kingdom (3). Esters of phenolic acids mainly belong to two distingui...

The hydrolase fold is one of the most versatile structures in the protein realm according to the diversity of sequences adopting such a three‐dimensional architecture. In the present study, we clarified the crystal structure of the carboxylesterase Cest‐2923 from the lactic acid bacterium Lactobacillus plantarum WCFS1 refined to 2.1 Å resolution, determined its main biochemical characteristics and also carried out an analysis of its associative behaviour in solution. We found that the versatility of a canonical α/β hydrolase fold, the basic framework of the crystal structure of Cest‐2923, also extends to its oligomeric behaviour in solution. Thus, we discovered that Cest‐2923 exhibits a pH‐dependent pleomorphic behaviour in solution involving monomers, canonical dimers and tetramers. Although, at neutral pH, the system is mainly shifted to dimeric species, under acidic conditions, tetrameric species predominate. Despite these tetramers resulting from the association of canonical dimers, as is commonly found in many other carboxylesterases from the hormone‐sensitive lipase family, they can be defined as ‘noncanonical’ because they represent a different association mode. We identified this same type of tetramer in the closest relative of Cest‐2923 that has been structurally characterized: the sugar hydrolase YeeB from Lactococcus lactis. The observed associative behaviour is consistent with the different crystallographic results for Cest‐2923 from structural genomics consortia. Finally, the presence of sulfate or acetate molecules (depending on the crystal form analysed) in the close vicinity of the nucleophile Ser116 allows us to identify interactions with the putative oxyanion hole and deduce the existence of hydrolytic activity within Cest‐2923 crystals. Structured digital abstract http://www.uniprot.org/uniprot/F9US10 and http://www.uniprot.org/uniprot/F9US10 http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407 by http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0114 (http://www.ebi.ac.uk/intact/interaction/EBI-8794536, http://www.ebi.ac.uk/intact/interaction/EBI-8794384)http://www.uniprot.org/uniprot/F9US10 and http://www.uniprot.org/uniprot/F9US10 http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407 by http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0028 (http://www.ebi.ac.uk/intact/interaction/EBI-8794545, http://www.ebi.ac.uk/intact/interaction/EBI-8794561) Database The atomic coordinates and structure factors have been deposited in the Protein Data Bank with accession numbers: http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4BZW for Cest‐2923 from native crystals not soaked with substrates (P6322 space group); http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4C01 for Cest‐2923 from crystals soaked with phenyl acetate (C2 space group); http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4BZZ for Cest‐2923 from crystals soaked with isopropenyl acetate (P622 space group).

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