Artificial Manganese Metalloenzymes with Laccase-like Activity: Design, Synthesis, and Characterization.

Laccase is an oxidase of great industrial interest due to its ability to catalyze oxidation processes of phenols and persistent organic pollutants. However, it is susceptible to denaturation at high temperatures, sensitive to pH, and unstable in the presence of high concentrations of solvents, which is a issue for industrial use. To solve this problem, this work develops the synthesis in an aqueous medium of a new Mn metalloenzyme with laccase oxidase mimetic catalytic activity. Geobacillus thermocatenulatus lipase (GTL) was used as a scaffold enzyme, mixed with a manganese salt at 50 °C in an aqueous medium. This leads to the in situ formation of manganese(IV) oxide nanowires that interact with the enzyme, yielding a GTL-Mn bionanohybrid. On the other hand, its oxidative activity was evaluated using the ABTS assay, obtaining a catalytic efficiency 300 times higher than that of Trametes versicolor laccase. This new Mn metalloenzyme was 2 times more stable at 40 °C, 3 times more stable in the presence of 10% acetonitrile, and 10 times more stable in 20% acetonitrile than Novozym 51003 laccase. Furthermore, the site-selective immobilized GTL-Mn showed a much higher stability than the soluble form. The oxidase-like activity of this Mn metalloenzyme was successfully demonstrated against other substrates, such as l-DOPA or phloridzin, in oligomerization reactions.

Structural and functional analysis of the key enzyme responsible for the degradation of ochratoxin A in the Alcaligenes genus.

The potential to degrade ochratoxin A (OTA), a highly poisonous mycotoxin, was investigated in cultures from Alcaligenes-type strains. Genome sequence analyses from different Alcaligenes species have permitted us to demonstrate a direct, causal link between the gene coding a known N-acyl-L-amino acid amidohydrolase from A. faecalis (AfOTH) and the OTA-degrading activity of this bacterium. In agreement with this finding, we found the gene coding AfOTH in two additional species included in the Alcaligenes genus, namely, A. pakistanensis, and A. aquatilis, which also degraded OTA. Notably, A. faecalis subsp. faecalis DSM 30030T was able to transform OTα, the product of OTA hydrolysis. AfOTH from A. faecalis subsp. phenolicus DSM 16503T was recombinantly over-produced and enzymatically characterized. AfOTH is a Zn2+-containing metalloenzyme that possesses structural features and conserved residues identified in the M20D family of enzymes. AfOTH is a tetramer in solution that shows both aminoacylase and carboxypeptidase activities. Using diverse potential substrates, namely, N-acetyl-L-amino acids and carbobenzyloxy-L-amino acids, a marked preference towards C-terminal Phe and Tyr residues could be deduced. The structural basis for this specificity has been determined by in silico molecular docking analyses. The amidase activity of AfOTH on C-terminal Phe residues structurally supports its OTA and OTB degradation activity.

Design of a gold nanoparticles site in an engineered lipase: an artificial metalloenzyme with enantioselective reductase-like activity.

The conjugation of gold complexes with proteins has proved to be interesting and effective in obtaining artificial metalloenzymes as catalysts with improved properties such as higher stability, activity and selectivity. However, the design and precise regulation of their structure as protein nanostructured forms level remains a challenge. Here, we have designed and constructed a gold nanoparticles-enzyme bioconjugate, by tailoring the in situ formation of gold nanoparticles (AuNPs) at two specific sites on the structure of an alkalophilic lipase from Geobacillus thermocatenulatus (GTL). For this purpose, two genetically modified variants of GTL were created by inserting a unique cysteine residue into the catalytic active site by replacing the active serine (GTL-114) and into the lid site (GTL-193). The enzyme, after a first protein-gold coordination, induced the in situ formation of AuNPs, generating a homogeneous artificial enzyme. The size and morphology of the nanoparticles in the AuNPs-enzyme conjugate have been controlled by specific pH conditions in synthesis and the specific protein region where they are formed. Reductase activity of all of them was confirmed in the hydrogenation of nitroarenes in aqueous media. The protein area seemed to be key for the AuNPs, with the best TOF values obtained for the bioconjugates with AuNPs in the lid site. Finally, the protein environment and the asymmetric properties of the AuNPs were tested in the reduction of acetophenone to 1-phenylethanol in aqueous medium at room temperature. A high reductive conversion and an enantiomeric excess of up to 39% towards (R)-1-phenylethanol was found using Au-Mt@GTL-114 pH 10 as a catalyst. Moderate enantioselectivity towards the opposite isomer was also observed using the Au-Mt@GTL-193 pH 10 conjugate.

A new and promiscuous α/ß hydrolase from Acinetobacter tandoii DSM 14970 T inactivates the mycotoxin ochratoxin A.

The presence of ochratoxin A (OTA) in food and feed represents a serious concern since it raises severe health implications. Bacterial strains of the Acinetobacter genus hydrolyse the amide bond of OTA yielding non-toxic OTα and L-ß-phenylalanine; in particular, the carboxypeptidase PJ15_1540 from Acinetobacter sp. neg1 has been identified as an OTA-degrading enzyme. Here, we describe the ability to transform OTA of cell-free protein extracts from Acinetobacter tandoii DSM 14970 T, a strain isolated from sludge plants, and also report on the finding of a new and promiscuous α/ß hydrolase (ABH), with close homologs highly distributed within the Acinetobacter genus. ABH from A. tandoii (AtABH) exhibited amidase activity against OTA and OTB mycotoxins, as well as against several carboxypeptidase substrates. The predicted structure of AtABH reveals an α/ß hydrolase core composed of a parallel, six-stranded ß-sheet, with a large cap domain similar to the marine esterase EprEst. Further biochemical analyses of AtABH reveal that it is an efficient esterase with a similar specificity profile as EprEst. Molecular docking studies rendered a consistent OTA-binding mode. We proposed a potential procedure for preparing new OTA-degrading enzymes starting from promiscuous α/ß hydrolases based on our results. KEY POINTS: • AtABH is a promiscuous αß hydrolase with both esterase and amidohydrolase activities • AtABH hydrolyses the amide bond of ochratoxin A rendering nontoxic OTα • Promiscuous αß hydrolases are a possible source of new OTA-degrading enzymes.

Dual 6Pß-Galactosidase/6Pß-Glucosidase GH1 Family for Lactose Metabolism in the Probiotic Bacterium Lactiplantibacillus plantarum WCFS1.

Intestinal lactic acid bacteria can help alleviate lactose maldigestion by promoting lactose hydrolysis in the small intestine. This study shows that protein extracts from probiotic bacterium Lactiplantibacillus plantarum WCFS1 possess two metabolic pathways for lactose metabolism, involving ß-galactosidase (ß-gal) and 6Pß-galactosidase (6Pß-gal) activities. As L. plantarum WCFS1 genome lacks a putative 6Pß-gal gene, the 11 GH1 family proteins, in which their 6Pß-glucosidase (6Pß-glc) activity was experimentally demonstrated,, were assayed for 6Pß-gal activity. Among them, only Lp_3525 (Pbg9) also exhibited a high 6Pß-gal activity. The sequence comparison of this dual 6Pß-gal/6Pß-glc GH1 protein to previously described dual GH1 proteins revealed that L. plantarum WCFS1 Lp_3525 belonged to a new group of dual 6Pß-gal/6Pß-glc GH1 proteins, as it possessed conserved residues and structural motifs mainly present in 6Pß-glc GH1 proteins. Finally, Lp_3525 exhibited, under intestinal conditions, an adequate 6Pß-gal activity with possible relevance for lactose maldigestion management.

Non-redundant functionality of Lactiplantibacillus plantarum phospho-ß-glucosidases revealed by carbohydrate utilization signatures associated to pbg2 and pbg4 gene mutants.

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

Production and Digestibility Studies of ß-Galactosyl Xylitol Derivatives Using Heterogeneous Catalysts of LacA ß-Galactosidase from Lactobacillus Plantarum WCFS1.

The synthesis of ß-galactosyl xylitol derivatives using immobilized LacA ß-galactosidase from Lactobacillus plantarum WCFS1 is presented. These compounds have the potential to replace traditional sugars by their properties as sweetener and taking the advantages of a low digestibility. The enzyme was immobilized on different supports, obtaining immobilized preparations with different activity and stability. The immobilization on agarose-IDA-Zn-CHO in the presence of galactose allowed for the conserving of 78% of the offered activity. This preparation was 3.8 times more stable than soluble. Since the enzyme has polyhistidine tags, this support allowed the immobilization, purification and stabilization in one step. The immobilized preparation was used in synthesis obtaining two main products and a total of around 68 g/L of ß-galactosyl xylitol derivatives and improving the synthesis/hydrolysis ratio by around 30% compared to that of the soluble enzyme. The catalyst was recycled 10 times, preserving an activity higher than 50%. The in vitro intestinal digestibility of the main ß-galactosyl xylitol derivatives was lower than that of lactose, being around 6 and 15% for the galacto-xylitol derivatives compared to 55% of lactose after 120 min of digestion. The optimal amount immobilized constitutes a very useful tool to synthetize ß-galactosyl xylitol derivatives since it can be used as a catalyst with high yield and being recycled for at least 10 more cycles.

A structurally unique Fusobacterium nucleatum tannase provides detoxicant activity against gallotannins and pathogen resistance.

Colorectal cancer pathogenesis and progression is associated with the presence of Fusobacterium nucleatum and the reduction of acetylated derivatives of spermidine, as well as dietary components such as tannin-rich foods. We show that a new tannase orthologue of F. nucleatum (TanBFnn ) has significant structural differences with its Lactobacillus plantarum counterpart affecting the flap covering the active site and the accessibility of substrates. Crystallographic and molecular dynamics analysis revealed binding of polyamines to a small cavity that connects the active site with the bulk solvent which interact with catalytically indispensable residues. As a result, spermidine and its derivatives, particularly N8 -acetylated spermidine, inhibit the hydrolytic activity of TanBFnn and increase the toxicity of gallotannins to F. nucleatum. Our results support a model in which the balance between the detoxicant activity of TanBFnn and the presence of metabolic inhibitors can dictate either conducive or unfavourable conditions for the survival of F. nucleatum.

Production of α-rhamnosidases from Lactobacillus plantarum WCFS1 and their role in deglycosylation of dietary flavonoids naringin and rutin.

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.

The commensal bacterium Lactiplantibacillus plantarum imprints innate memory-like responses in mononuclear phagocytes.

Gut microbiota is a constant source of antigens and stimuli to which the resident immune system has developed tolerance. However, the mechanisms by which mononuclear phagocytes, specifically monocytes/macrophages, cope with these usually pro-inflammatory signals are poorly understood. Here, we show that innate immune memory promotes anti-inflammatory homeostasis, using as model strains of the commensal bacterium Lactiplantibacillus plantarum. Priming of monocytes/macrophages with bacteria, especially in its live form, enhances bacterial intracellular survival and decreases the release of pro-inflammatory signals to the environment, with lower production of TNF and higher levels of IL-10. Analysis of the transcriptomic landscape of these cells shows downregulation of pathways associated with the production of reactive oxygen species (ROS) and the release of cytokines, chemokines and antimicrobial peptides. Indeed, the induction of ROS prevents memory-induced bacterial survival. In addition, there is a dysregulation in gene expression of several metabolic pathways leading to decreased glycolytic and respiratory rates in memory cells. These data support commensal microbe-specific metabolic changes in innate immune memory cells that might contribute to homeostasis in the gut.

Geranyl Functionalized Materials for Site-Specific Co-Immobilization of Proteins.

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.

Pd-Oxazolone complexes conjugated to an engineered enzyme: improving fluorescence and catalytic properties.

Different Pd-complexes containing orthometallated push-pull oxazolones were inserted by supramolecular Pd-amino acid coordination on two genetically engineered modified variants of the thermoalkalophilic Geobacillus thermocatenolatus lipase (GTL). Pd-lipase conjugation was performed on the solid phase in the previously immobilized form of GTL under mild conditions, and soluble conjugated Pd-GTL complexes were obtained by simply desorbing by washing with an acetonitrile aqueous solution. Three different Pd complexes were incorporated into two different genetically modified enzyme variants, one containing all the natural cysteine residues changed to serine residues, and another variant including an additional Cys mutation directly in the catalytic serine (Ser114Cys). The new Pd-enzyme conjugates were fluorescent even at ppm concentrations, while under the same conditions free Pd complexes did not show fluorescence at all. The Pd conjugation with the enzyme extremely increases the catalytic profile of the corresponding Pd complex from 200 to almost 1000-fold in the hydrogenation of arenes in aqueous media, achieving in the case of GTL conjugated with orthopalladated 4a an outstanding TOF value of 27 428 min-1. Also the applicability of GTL-C114 conjugated with orthopalladated 4b in a site-selective C-H activation reaction under mild conditions has been demonstrated. Therefore, the Pd incorporation into the enzyme produces a highly stable conjugate, and improves remarkably the catalytic activity and selectivity, as well as the fluorescence intensity, of the Pd complexes.

The use of Lactobacillus plantarum esterase genes: a biotechnological strategy to increase the bioavailability of dietary phenolic compounds in lactic acid bacteria.

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.

Biosynthesis of Nondigestible Galactose-Containing Hetero-oligosaccharides by Lactobacillus plantarum WCFS1 MelA α-Galactosidase.

This work describes the high capacity of MelA α-galactosidase from Lactobacillus plantarum WCFS1 to transfer galactosyl residues from melibiose to the C6-hydroxyl group of disaccharide-acceptors with ß-linkages (lactulose, lactose, and cellobiose) or α-linkages (isomaltulose and isomaltose) to produce novel galactose-containing hetero-oligosaccharides (HOS). A comprehensive nuclear magnetic resonance characterization of the transfer products derived from melibiose:lactulose reaction mixtures revealed the biosynthesis of α-d-galactopyranosyl-(1 → 6)-ß-d-galactopyranosyl-(1 → 4)-ß-d-fructose as the main component as well as the presence of α-d-galactopyranosyl-(1 → 3)-ß-d-galactopyranosyl-(1 → 4)-ß-d-fructose and α-d-galactopyranosyl-(1 → 6)-α-d-galactopyranosyl-(1 → 6)-ß-d-galactopyranosyl-(1 → 4)-ß-d-fructose. Melibiose-derived α-galactooligosaccharides (α-GOS), manninotriose and verbascotetraose, were also simultaneously synthesized. An in vitro assessment of the intestinal digestibility of the novel biosynthesized HOS revealed a high resistance of α-galactosides derived from lactulose, lactose, cellobiose, and isomaltulose. According to the evidence gathered for conventional α-GOS and certain disaccharides used as acceptors in this work, these novel nondigestible α-galactosides could be potential candidates to selectively modulate the gut microbiota composition, among other applications, such as low-calorie food ingredients.

Molecular Responses of Lactobacilli to Plant Phenolic Compounds: A Comparative Review of the Mechanisms Involved.

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

Hydrolysis of Lactose and Transglycosylation of Selected Sugar Alcohols by LacA ß-Galactosidase from Lactobacillus plantarum WCFS1.

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

Unravelling the carbohydrate specificity of MelA from Lactobacillus plantarum WCFS1: An α-galactosidase displaying regioselective transgalactosylation.

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 â†’ 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.

Oleuropein Transcriptionally Primes Lactobacillus plantarum to Interact With Plant Hosts.

Oleuropein (OLE) is a secoiridoid unique to Oleaceae known to play a role in the plant-herbivore interaction. However, it is not clear how this molecule is induced to mediate plant responses to microbes and how microbes, in turn, withstand with OLE. To better understand how OLE affects the plant-microbe interaction, the contribution of differential gene expression in the adaptation to OLE was characterized by whole genome transcriptional profiling in Lactobacillus plantarum, a bacterium associated to the olive. OLE downregulated functions associated to rapid growth, remodeled membrane phospholipid biosynthesis pathways and markedly repressed the expression of several ABC transporters from L. plantarum. Genes encoding the plantaricin and lamABDCA quorum-sensing (QS) systems were down-regulated indicating the potential of OLE as a QS-antagonist. Notably, OLE diminished the expression of a set of genes encoding inmunomodulatory components and reoriented metabolic pathways to increase protein acetylation, probably to attenuate plant immunity. Responses were also triggered to repress the transport of acetoin and to buffer reactive oxygen species accumulation, two signals involved in plant development. The results suggest that OLE could act as a signaling molecule in the plant-microbe interaction and facilitate the accommodation of beneficial microbes such as L. plantarum by the plant host, via controlled expression of bacterial molecular players involved in this reciprocal interplay.

Unravelling the diversity of glycoside hydrolase family 13 α-amylases from Lactobacillus plantarum WCFS1.

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.

Bacterial tannases: classification and biochemical properties.

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