Identification of a highly active tannase enzyme from the oral pathogen Fusobacterium nucleatum subsp. polymorphum

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. Electronic supplementary material The online version of this article (10.1186/s12934-018-0880-4) contains supplementary material, which is available to authorized users.


Background
Tannins are high molecular weight secondary phenolic metabolites of plant origin that have been traditionally considered antinutrients due to their capacity to bind and precipitate protein [1,2]. This group of chemicals can be found in tea, wine, berries, fruits and chocolate among other dietary components. They are toxic compounds for a variety of microorganisms because of their protein and iron binding capacity, and may interfere with many biological processes that are essential for their growth [1]. In turn, some microorganisms have developed strategies to overcome tannin toxicity, including the presence of genes in their genomes encoding degrading enzymes such as tannase enzymes [3]. Initially, tannases, also known as tannin acyl hydrolases (EC 3.1.1.20), were described in fungi and studied primarily because of their industrial use in processes related to food detannification

Open Access
Microbial Cell Factories *Correspondence: janguita@cicbiogune.es; hrodriguez@cicbiogune.es † Julen Tomás-Cortázar and Laura Plaza-Vinuesa contributed equally to this work 1 Macrophage and Tick Vaccine Laboratory, CIC bioGUNE, Derio, Bizkaia, Spain Full list of author information is available at the end of the article and the removal of pollutants, in the leather industry or for the production of gallic acid, which is an important intermediate in the synthesis of the antibiotic trimethoprim [3,4]. Recently, tannases have also been isolated from bacteria that populate environments rich in vegetable content, although just a few of the genes encoding bacterial tannases have been described and even fewer have been characterized biochemically. The most studied tannases are present in bacteria isolated from the rumen, gut microbiota or soils with abundant vegetation [5][6][7][8][9][10][11]. Two different types of tannases have been described so far that are encoded by two different genes. Extracellular tannases (encoded by the tanA gene) contain a signal peptide and a molecular size of around 66 kDa. These include TanA Sl from Staphylococcus lugdunensis [5,9,12,13], TanA Lp from Lactobacillus plantarum [8], and TanA Sg from Streptococcus gallolyticus [14]. In addition, 50 kDa intracellular bacterial tannases (encoded by tanB genes) have also been described. These proteins are found in L. plantarum (TanB Lp ) [6,15] or S. gallolyticus (TanB Sg ) [7]. Recently, an extracellular tannase that is nevertheless encoded by a tanB gene has been described in Streptomyces sviceus (TanB Ss ) [11].
The gut and the oral cavity harbor distinctive populations of bacteria permanently exposed to a vast array of chemical compounds present in food, including tannins. The human microbiota is a massive and largely unexplored source of enzymes with new and/or improved activities [16]; however, limited research has been performed to identify microbes capable of degrading tannins within the inhabitants of the human body. Although bacteria that contain enzymes with tannase activity, such as L. plantarum, S. gallolyticus, or S. lugdunensis, have been described in the human gastrointestinal tract [9,[17][18][19], many questions remain about the oral metabolism of food tannins. As the oral cavity is continuously exposed to tannins, we hypothesized that oral bacteria might harbor tannin-degrading genes in their genomes. Previously, a tannase (TanA Ap ) from Atopobium parvulum, a species abundant in the oral cavity, was characterized [20]. This protein exhibited the lowest specific activity among bacterial tannases and was unable to hydrolyze complex tannins. Therefore, the biochemical properties of TanA Ap discarded its role in the breakdown of complex food tannins. Herein, we have identified and characterized the first tannase enzyme described within the genus Fusobacterium and overall, from a pathogenic bacterium. The study of its biochemical properties and the substrates among different relevant tannin derivatives present in food demonstrate that it is among the most active tannases described so far possessing a wide range of substrate specificity that includes several derivatives of gallic acid, protocatechuic acid and complex tannins.

Presence of a putative tannase in members of the genus Fusobacterium
A search for L. plantarum tannase (TanB Lp ) homologues was performed in common oral bacteria genera using NCBI blastx. Among the hits, a gene from Fusobacterium nucleatum subsp. polymorphum (annotated as a hypothetical protein) showed 44% identity to TanB Lp (GMHT-1603-MONOMER from BioCyc database) [21]. Because of the identity, we annotated the gene as tanB Fnp and the protein that it encodes as TanB Fnp . In addition, the CDD web tool revealed that TanB Fnp presented domains conserved among bacterial tannases including the essential amino acids for hydrolytic-esterase activity previously described as the active site of the protein [22]. Alignment of the whole amino acid sequence and a partial alignment showing just key residues are showed in Fig. 1. The presence of these key residues identified using Python's WebLogo package (Fig. 1b) suggested that the F. nucleatum subsp. polymorphum gene product is a relevant candidate to have tannase activity. We also performed a phylogenetic analysis of this gene with other tannases previously identified in order to get further information about their proximity. The dendrogram in Fig. 2 shows that TanB Fnp is more similar to L. plantarum TanB Lp tannase than to all the bacterial tannases previously described. We also analyzed the distribution of orthologs of the F. polymorphum tannase in species that belong to the Fusobacterium genus and other oral bacteria. Orthologs for TanB Fnp were found in all F. nucleatum subspecies with the exception of the subspecies fusiforme as well as in other Fusobacterium species related to oral and gut diseases, such as F. periodonticum, F. necrophorum, F. massiliense and F. hwasooki. Strikingly, more than 30% of the total number of TanB Fnp orthologs detected were found among oral bacteria species, including the most prevalent genera identified in the human oral microbiota (Prevotella, Neisseria, Streptococcus, Fusobacterium and Haemophilus) [23] (Additional file 1: Table S1).

TanB Fnp is among the most active bacterial tannases identified to date
The Fusobacterium putative tannase TanB Fnp is a 58 kDa protein with an alkaline isoelectric point (theoretical pI, 8.9) and a predicted signal peptide comprising the 20 initial residues. The presence of a signal peptide is not a common characteristic among TanB type tannases. In order to avoid solubility issues, the gene was cloned without the signal peptide into the pHis-parallels II expression vector and purified using His-affinity chromatography combined with a gel filtration step (Fig. 3). In order to address whether TanB Fnp was, in fact, a tannase enzyme, a colorimetric assay was performed. Rhodanine assay [24] indirectly detects tannase activity over methyl gallate by measuring colorimetrically at 520 nm a b Fig. 1 Comparison of TanB Fnp with previously described bacterial tannases. a Alignment of the whole protein sequences of bacterial tannases showing conserved regions. b Alignment of conserved motifs in bacterial tannases and sequence logos depicting the consensus sequence and diversity of bacterial tannase sequences. The sequences corresponding to those domains predicted to have hydrolase activity and with the highest conservation scores were used to identify a consensus sequence. The color scheme is defined by hydrophobicity scale (amino acids representation default), where each color corresponds to the following code: hydrophilic residues (RKDENQ) in blue, neutral residues (SGHTAP) in green, and hydrophobic (YVMCLFIW) in black the binding of the vicinal hydroxilic groups of the reaction product, gallic acid, with rhodanine (see "Methods"). Rhodanine reacts specifically with gallic acid while it does not bind other phenol compounds. Once the activity was confirmed, the absolute activity of TanB Fnp was measured, using Lactobacillus plantarum TanB Lp as a reference. The activity of TanB Fnp using methyl gallate as a substrate was one of the highest described to date in bacteria (699 U/mg) being equivalent to a recently isolated Streptococcus lugdunensis enzyme (716 U/mg), and 76.5% higher than TanB Lp , the reference used in the same experiment (395.9 U/mg). TanB Fnp activity was more than 20% higher than the one described for Streptococcus gallolyticus, TanB Sg [7] (Fig. 4d).

Biochemical characterization of TanB Fnp
We then determined the biochemical properties of TanB Fnp using the purified protein. Its optimum activity pH was measured at 30 °C in 50 mM phosphate buffer. Figure 4a shows an optimum pH for TanB Fnp of 7 and suboptimal activity of the enzyme at pH values ranging from 6 to 8, although it still retained around 80% of the maximal activity. The optimal activity temperature was determined incubating the protein in 50 mM phosphate buffer pH 6.5. TanB Fnp showed the highest activity at 55 °C, albeit it retained around 80% of the maximal activity at all temperatures tested between 22 and 55 °C (Fig. 4b). The thermal stability of the enzyme was also determined by pre-incubation at different temperatures ranging from 22 to 65 °C for increasing lengths of time up to 20 h, followed by activity determination. Surprisingly, pre-incubation of the enzyme between 22 and 45 °C did not substantially affect the activity of the enzyme, maintaining methyl gallate hydrolysis rates above 60% of the maximum activity and in some cases, increasing the enzymatic activity. On the contrary, incubation at temperatures higher than 45 °C induced a dramatic decrease in TanB Fnp activity after 30 min of incubation (Fig. 4c).
We also tested the influence of distinct additives on TanB Fnp activity ( Table 1). The addition of HgCl 2 and β-mercaptoethanol abolished the activity of the enzyme whereas the rest of the compounds tested increased the

Substrate specificity of TanB Fnp
In order to identify relevant food substrates for TanB Fnp , the purified enzyme was incubated with different esters of phenolic acids and the reaction products were then analyzed using high-performance liquid chromatography coupled with a diode array detection unit (HPLC-DAD). As a control, all the reactions were also performed and analyzed in the absence of the enzyme (see "Methods"). TanB Fnp hydrolyzed several simple esters of gallic acid (3,4,5-trihydroxybenzoic acid) regardless of the length of their aliphatic alcohol. Among this group of compounds, methyl gallate, ethyl gallate, propyl gallate and lauryl gallate (Fig. 5a) were transformed into gallic acid by TanB Fnp . Other substrates, including polyphenolic esters of gallic acid such as gallocatechin gallate and epigallocatechin gallate were also hydrolyzed (Fig. 5b). The ability of the enzyme to break complex natural tannins was also studied using tannic acid as a substrate. This complex phenolic compound was almost entirely hydrolyzed by TanB Fnp , generating a simpler mix of compounds with gallic acid as the predominant product (Fig. 5c). Finally, esters of protocatechuic acid such as ethyl 3,4-dihydroxybenzoate and ethyl 3,5-dihydroxybenzoate were also degraded by the enzyme, being both converted into protocatechuic acid (Fig. 5c). These results demonstrated that TanB Fnp is active against a wide spectrum of gallic and protocatechuic acid-derived substrates.  Table 2 shows the battery of compounds tested in this study and whether they were hydrolyzed by the enzyme.

Discussion
The search for new tannase enzymes with improved activity and new substrate specificities constitutes a permanent focus for industrial microbiology research due to their extensive use in the food and pharmaceutical industries. Indeed, the search for enzymes with tannase activity has recently been expanded to the human microbial population. Herein, we describe a new tannase enzyme from the oral pathogen F. nucleatum susbp. polymorphum. Oral bacteria need to have extensive metabolic resources to face the presence of food components with antimicrobial properties that they may encounter. We previously described a tannase from A. parvulum, an inhabitant of the human oral cavity [20]. However, this tannase (TanA Ap ) possessed low specific activity and was unable to hydrolyze complex tannins. Therefore, it is unlikely that this tannase contributes to tannin breakdown. Because the presence of enzymes with tannase activity is one of the preferred mechanisms to overcome phenolic-related stress, we sought and identified a homologous protein of the tannase from L. plantarum, TanB Lp , within the Fusobacterium genus. Phylogenetically, TanB Fnp shows a closer relationship with TanB Lp than with all other bacterial tannases. TanB Lp has been previously associated with a cluster of tannases unrelated to those of fungal origin [8]. Therefore, TanB Fnp might be included within the same cluster of bacterial tannindegrading enzymes.
The biochemical characterization of TanB Fnp reveals that it is an enzyme with potential industrial applications. Its activity against methyl gallate is among the highest described in bacteria so far and considerably higher than other tannases previously reported [7,8,10,11,15,20]. Other biotechnological features that are important for its potential industrial application include its temperature range of activity. The optimal temperature of most tannases varies between 30 and 40 °C [4]. Strikingly, TanB Fnp is highly active in a much wider range of temperatures, from 22 to 60 °C, with a maximum activity peak at 60 °C, similar to S. gallolyticus (TanB Sg ) and S. sviceus tannases (TanB Ss ) [7,11]. Accordingly, TanB Fnp thermal stability is much higher than that of all previously described tannases. Similar to TanB Sg , TanB Fnp is able to keep high activity rates after it has been exposed for long time intervals to temperatures ranging from 22 to 45 °C while most bacterial tannases suffer a deep decrease in their activity after long exposures to temperatures equal to or higher than 37 °C. As previously suggested [11], it is possible that for extracellular tannases, such as TanB Fnp , a better adaptability to the harsh extracellular environment has induced the development of more resistant enzymes. Both the high performance temperature and its thermal resistance to unfolding or denaturalization in the absence of substrate are key features that support TanB Fnp biotechnological use. These characteristics may likely improve its interaction with substrates, favor high mass transfer rates, and lower the risk of contamination [25].
Like all bacterial tannases characterized to date, TanB Fnp showed the highest activity rates at pH between 6 and 8, which overlaps the pH of the oral cavity (between 6.7 and 7.3). The neutral pH range of activity seems to be a common characteristic of bacterial tannases in contrast with those of fungal origin that present activity peaks under acidic conditions [26].
The search for enzymes with new activities is also an important target in tannase research due to the high variety of tannin substrates. We performed a substrate characterization using 27 different compounds from different  [7,8], TanB Fnp is capable of degrading esters of phenolic acids with long chain alcohols such as lauryl gallate. The hydrolyzing activity of TanB Fnp against longer esters points to a markedly different substrate-binding site in this enzyme that would permit the access of these bulkier compounds. Fusobacterium nucleatum subsp. polymorphum is an inhabitant of the human oral cavity frequently isolated from dental plaque biofilms [27]. As a pathogen, it has been repeatedly associated with peridontitis and extraoral infections including preterm births and colorectal cancer [28]. However, little is known about the metabolic arsenal that allows them to thrive in these environments. The extracellular tannase identified in F. nucleatum susps. polymorphum, TanB Fnp , might provide an ecological advantage to bacteria thriving in a niche permanently exposed to phenolic stress from food sources. The enzyme is still highly active (80% of its optimal activity) at body temperature and at pH usually found within the human body. Vegetable food residues are permanently deposited in the oral cavity as a consequence of food intake. An extracellular tannase could be an important survival factor for F. nucleatum with a dual role during bacterial colonization of teeth: the detoxification of toxic compounds and the provision of a source of sugar moieties resulting from the hydrolyzation of complex tannins. The wide distribution of tannase enzymes among oral inhabitants in comparison to microbial species in other niches suggests that its presence may constitute an adaptative advantage to compete in an environment that is permanently receiving tannins from diet sources. Therefore, TanB Fnp may constitute a putative virulence factor for F. nucleatum and a potential target of therapeutic intervention for this pathogen.
The description of TanB Fnp increases our knowledge about tannin breakdown in the human gastrointestinal tract. Oral and intestinal tannases could contribute to tannin digestion. At least five different species of oral or intestinal bacteria have been described to possess active tannases, including A. parvulum, L. plantarum, S. gallolyticus, S. lugdunensis and, in this work, Fusobacterium nucleatum subsp. polymorphum. Further testing would be required to define the metabolism of these phenolic compounds comprehensively. In particular, the activity of the complex communities of microorganisms present in all parts of the human digestive tract would need to be examined. Moreover, as tannase-producing bacteria have been identified in several human cancer microbiomes [29], the study of the association between dietary tannin intake and tumor recurrence or regression, may be critical in understanding the role of gut bacteria on the anticancer effects of dietary polyphenols.

Conclusions
In this work, we describe an enzyme with tannase activity in the oral pathogen Fusobacterium nucleatum subsp. polymorphum. TanB FNP is one of the most active tannases described to date. This fact combined with an extraordinary thermal stability and a wide range of temperature activity makes TanB FNP a suitable candidate for industrial applications. Moreover, since F. nucleatum subsp. polymorphum is a pathogen associated with oral and extraoral diseases our research increases the knowledge about a putative niche adaptation determinant that might be involved in virulence associated with the transformation of diet antimicrobial compounds.

Bacterial strains and growth conditions
Escherichia coli strains DH5α and BL21(DE3) were used as transformation and expression hosts, respectively, for the pHISp-fusotan vector. The bacteria were grown in LB medium containing ampicillin (100 µg/mL) at 37 °C with agitation.

Gene cloning
Genomic DNA from F. nucleatum subsp. polymorphum Strain F0401 was obtained from BEI Resources Repository. Standard molecular biology procedures [30] were followed to clone the tannase gene avoiding the signal peptide of the protein. The gene was PCR-amplified using phusion hot start II polymerase (Thermo Fisher Scientific, Waltham, MA) with primers Fusotannase-f (5′-CGC CAT GGG CGT AAA AAA TGA GTA TGA TT-3′) and Fusotannase-r (5′-CGG TCG ACT TAT TTT TTT ACA ACA CCA TC-3′). The PCR product was purified using a PCR purification kit (Thermo Fisher Scientific). The 1.5 kb PCR product and the vector pHis parallel 2 (Addgene) were digested with NcoI and SalI and then ligated for 16 h using T4 DNA ligase (Promega, Madison, WI). The ligation mixture was transformed in DH5α cells and positive colonies were verified by colony PCR and sequencing. Positive plasmids (pHISp-fusotan) were finally transformed into Escherichia coli BL21(DE3) cells for protein production.

Protein production and purification
Sequence-confirmed TanB Fnp clones in E. coli BL21(DE3) were grown in Luria-Bertani medium supplemented with ampicillin and induced with 1 mM IPTG for 16 h at 20 °C. The His-tag fusion protein was then purified by nickel affinity chromatography (GE Healthcare, Uppsala, Sweden) and eluted in 20 mM Tris, pH 7.5 with 150 mM NaCl and 250 mM Imidazole. For a second purification step using gel filtration chromatography, fractions containing TanB Fnp identified by SDS-PAGE were pooled, concentrated and loaded onto a HiLoad 10/300 GL Superdex 75 column (GE Healthcare) pre-equilibrated in 20 mM Tris pH 7.5; 150 mM NaCl, using an AKTA chromatography system (GE Healthcare). Fractions with the protein of interest were pooled and the protein was concentrated and stored at − 80 °C until its use. Protein concentration was determined using the BCA protein assay kit (Thermo Fisher Scientific).

Determination of tannase activity
The generation of gallic acid in hydrolysis reactions was determined in triplicate with the following assay: TanB Fnp (10 μg) in 700 μL of 50 mM phosphate buffer, pH 6.5, was incubated with 40 μL of 25 mM methyl gallate (1 mM final concentration) for 5 min at 37 °C. After incubation, 150 μL of a methanolic rhodanine solution (0.667% rhodanine in 100% methanol) was added to the reaction mixture. After 5 min of incubation at 30 °C, 100 μL of 0.5 M KOH was added and the absorbance at 520 nm was measured on a spectrophotometer. A standard curve using gallic acid concentrations ranging from 0.125 to 1 mM was prepared. One unit of tannase activity was defined as the amount of enzyme required to release 1 μmol of gallic acid per minute under standard reaction conditions.

Biochemical characterization
Activities of Tan Fnp from F. nucleatum subsp. polymorphum F401 were measured at 4,20,30,37,45,55, and 65 °C to determine the optimal temperature for enzymatic activity. The optimum pH value for tannase activity was determined by studying its pH dependence within the pH range between 3 and 10. The buffers used were: acetic acid-sodium acetate buffer for pH 3-5, citric acidsodium citrate for pH 6, sodium phosphate for pH 7, Tris-HCl for pH 8, glycine-NaOH for pH 9, and sodium carbonate-bicarbonate for pH 10. A 100 mM concentration was used in all the buffers. The rhodanine assay was used for the optimal pH characterization of TanB Fnp [8]. Since the rhodanine-gallic acid complex forms only under basic conditions, after the enzymatic degradation of methyl gallate, 100 μL of 0.5 M KOH was added to the reaction mixture to ensure that the same pH value (pH 11) was achieved in all samples assayed.
To test the effects of metals and ions on the activity of TanB Fnp , the enzymatic activity was measured in the presence of different additives at a final concentration of 1 mM. The additives analyzed were MgCl 2 , KCl, CaCl 2 , HgCl 2 , ZnCl 2 , Triton X-100, urea, Tween 80, EDTA, dimethyl sulfoxide (DMSO) and β-mercaptoethanol. All the determinations were done in triplicate.

Bioinformatic analyses
The sequences of tanases analyzed in this work were inspected for conserved functional domains with the CDD web tool [31]. Then, the region harboring the functional domain related to the tanase activity was extracted. These sequences were the input for a Multiple Sequence Analysis using CLUSTAL omega [32], in order to identify conserved amino acid patterns among them, and use it as input for the phylogenetic analysis carried out by the Phylogeny.fr web-service [33] including the following steps: the CLUSTAL omega alignment was used as input, followed by the removal of poorly aligned/gapped regions using Gblocks v0.91b [34] with default settings for both tools. Phylogenetic trees were reconstructed using the maximum likelihood method with the PhyML program v3.0 [35] using the WAG substitution model and 100 bootstrap replicates for inner branch accuracy. The graphical representation and edition of the phylogenetic tree was performed with Tree-Dyn v198.3 [36]. Sequence logos were obtained using Python's WebLogo package [37] using the CLUSTAL omega's alignment file as input. The scale of the logo was measured in bits, which are units of measure with a precise thermodynamic relationship to energy. Display error bars indicate an approximate Bayesian 95% confidence interval.
In order to study the distribution of tannase ortholog genes among Fusobacterium spp. (in dental plaque and gut microflora) and other oral bacteria, we performed a BLASTp [38] search against the non-redundant database limited to bacteria taxa. Results were filtered by e-value (< 1e−20). Those with a query coverage > 50% and sequence identity > 20% were retained as putative orthologs (Additional file 1: Table S1).