Biochemical and molecular characterization of the endo-acting 6O-methyl-porphyranase BpGH16C (Bacple_01703)

The gene Bacple_01703 encodes a glycoside hydrolase of the GH16 family (BpGH16C), which includes characterized agarases in sub-families GH16_15-16, and β-porphyranases in sub-families GH16_11-12 [26]. BpGH16C was classified in the GH16_14 sub-family that contains one characterized β-agarase (Vibrio sp. strain PO-303, BAF62129.1) [27], one endo-6O-methyl-β-porphyranase (Wenyingzhuangia fucanilytica, WP_068825734.1) [28] and one β-porphyranase observed in the red algal Chondrus crispus [29]. We assayed BpGH16C on various marine polysaccharides including carrageenan, agarose and porphyran. Only porphyran was degraded, leading to the production of a series of oligosaccharides characteristic of endo-acting glycoside hydrolase (Fig 1A). The structural characterization by NMR of the end-products purified by chromatography [30] revealed the occurrence of a methyl group on the β-linked D-galactose residue (Fig 1B), demonstrating that BpGH16C is an endo-6O-methyl-β-porphyranase able to accommodate the methyl group of porphyran in its active site. For comparison, we have examined the substrate specificities of one another predicted GH16_14 endo-6O-methyl-β-porphyranase from Paraglaciecola atlantica T6c (Patl_0824, ABG39352.1) presenting 51.1% identity with the BpGH16C and one GH16_12 β-porphyranase (Patl_0805, ABG39333.1) homologous to BpGH16B. Analyses of the degradation products confirmed the different porphyranase specificities (Fig 1A).

We determined the crystal structure of BpGH16C at 1.9 Å resolution (PDB ID 8EP4) (Fig 2A). The enzyme has a β-sandwich jelly-roll fold with two stacking antiparallel β-sheets typical of proteins from GH16 family [26] and like in most GH16, a Ca²⁺ is bound on the convex side of the β-sandwich [31]. One molecule of Hepes from the crystallization buffer bound in the center of the cleft, with the 2-hydroxyethyl end buried into the molecule interior and the sulfate group exposed on protein surface (Fig 2A). To get a better understanding of substrate binding and catalysis, we crystallized the E145L inactive mutant with the tetrasaccharide L-α-6O-sulfate-Gal-(1 → 3)-D-β-Gal-(1 → 4)-L-α-6O-sulfate-Gal-(1 → 3)-D-a/β-Gal and determined its structure at 1.8 Å resolution (PDB ID 8EW1). An electron density was present within the groove in all the three independent molecules in the asymmetric unit fitted a D-β-Gal-(1 → 4)-L-α-6O-sulfate-Gal-(1 → 3)-D-β-Gal trisaccharide (Fig 2B). The electron density for the D-Gal on the nonreducing end is weaker than for the first two residues and no density is observed for the fourth residue indicating its mobility in the crystal. The residues correspond to the −1, −2 and −3 positions according to the standard nomenclature [32].

The trisaccharide sits edge-on in the groove with C5 substituent of the ring in the −1 site directed toward the bottom of the cleft while O1 and O2 hydroxyls point toward the solvent. Trp134 stacks against hydrophobic side of the −1 residue while Arg67 and Glu238 hydrogen bond to the O4 hydroxyl. Residue in the −2 subsite is stacked between Trp64 and the edges of Trp143 and Phe169. Finally, the sulfate group of the −2 residue is hydrogen bonded to Asn61, Ser136 and Asn234 through a bridging water molecule. The D-Gal on the non-reducing end (−3 site) has somewhat different orientation in molecules A/B and C and makes only one bridging hydrogen bond through a bridging water (Fig 2C). The structure of the complex with a trisaccharide adds also to our understanding of the catalytic mechanism. Glu150 is indeed the closest to the O1 hydroxyl that formed the bond to the next sugar eliminated by hydrolysis, while Glu145 approaches from the opposite side of the ring relative the O1 hydroxyl (Fig 3C) and is likely helping in proper orientation of the substrate. Asp147 is directed to the bridging O1 and might be helping in catalysis.

Structural alignment of BpGH16C with three other GH16 porphyranases: porphyranase A (PDB ID 3ILF) and porphyranase B from Zobellia galactanivorans (PDB ID 3JUU) [9] and another porphyranase from B. plebeius (PDB ID 4AWD) [16] shows that the Gly132 is replaced by either a Ser or a Thr, making this pocket too small to accommodate an additional methyl group on the C6-O (Fig 2D). Sequence alignment of β-porphyranases with BpGH16C and the two other characterized 6O-methyl-β-porphyranases: Patl_0824 of (P. atlantica T6c, this study) and AXE80_06940 (W. fucanilytica) [28] confirmed that the replacement of the Ser/Thr by Gly was correlated to the accommodation of the methyl group in the −1 sub-site of the catalytic site (S2 Fig). The BpGH16C is the first crystallized 6-OMe-porphyranase that tolerates the 6-methyl group on the galactose ring. Indeed, there is an extra space in the active site at the end of the pocket that accommodates C6-OH, with its bottom formed by Gly132 (Fig 2E).

Catabolism of oligo-methyl-porphyrans

Following the same strategy reported by Robb and co-workers [17], we have incubated the methylated oligosaccharides with the BpS1_11 sulfatase, the BpGH29 α-L-galactosidasee and the predicted BpGH2Cβ-D-galactosidase. Purified methylated and non-methylated oligo-porphyrans were incubated with the BpS1_11 sulfatase and analyses of the products by ¹H NMR showed a strong chemical shift (about 0.1 ppm) of the signal attributed to the proton at position 6 of the galactose located at the non-reducing end, demonstrating the removal of the sulfate ester group independently of the degree of methylation of the oligosaccharides (S3 Fig).

Crystal structure of the BuS1_11 sulfatase from B. uniformis complexed with a di-saccharides [18] and the B. plebeius sulfatase BpS1_11 on its own (this study, PDB ID 7SNJ, 1.64 Å resolution) and complexed with a tetrasaccharide (this study, PDB ID 7SNO, 2.1 Å resolution) revealed that there is room in the active site to accommodate oligo-methyl-porphyrans (S4 Fig). The porphyran sulfatase BpS1_11 belongs to the formyl-glycine dependent sulfatases, requiring maturation of a cysteine or a serine into the formyl-glycine residue as an active catalytic residue. A large N-terminal domain of BS1_11 contains active site residues and forms main part of the substrate binding site. A smaller C-terminal domain completes the substrate binding site.

To map the details of substrate binding, we determined the structure of H214N inactive mutant with L-α-6O-sulfate-Gal-(1 → 3)-D-β-Gal-(1 → 4)-L-α-6O-sulfate-Gal-(1 → 3)-D-a/β-Gal tetrasaccharide substrate (PDB ID 7SNO). The electron density map clearly showed the entire tetrasaccharide bound to BpS1_11 (H214N). Following the recently proposed nomenclature [33], the tetrasaccharide binds to the 0, + 1, + 2 and +3 positions (S4 Fig). The 6S-L-Gal in position 0, which the sulfate is removed, has all three hydroxyl groups hydrogen bonded to the protein side chains: O2 to Arg271, O3 to Asp215 and Arg271, O3 to Asp345 and Arg347. The 6-sulfate group is facing Ser83 (formyl-glycine in mature enzyme) and one of its oxygens is a ligand to the Ca²⁺ ion. D-Gal in +1 position hydrogen bonds C6OH to NE2 of His133 and its O2 is bonded to the O2 of L-Gal (position 0) through a bridging water molecule. The + 2 6S-L-Gal makes only one contact, between the 6-sulfate oxygen and His428. The + 3 D-Gal extends far from the binding site and makes no contacts with the protein. However, in the crystal, this sugar hydrogen bonds from O1 and O4 as well as O5 within the ring to the guanidino group of Arg68 from a symmetry-related molecule. These interactions stabilize the + 2 and +3 sugars in the crystal. Importantly, the 6-O group of +1 D-Gal is not tightly constraint by the enzyme and there is sufficient space for the additional methyl group present in the 6-O-Me-D-Gal (S4 Fig).

We confirmed the exo-based cycle of porphyran depolymerization [17] using tetra-saccharides as starting substrate (Fig 3, S1 Table). After desulfation of the tetra-saccharides, BpGH29 exo-α-L-galactosidase was active on the methylated and non-methylated substrate demonstrating that the methyl group did not hinder the activity of the enzyme. Independently of Robb and co-workers [17], we have determined the structure of BpGH29 (PDB ID 7SNK). The superposition with their structure (PDB ID 7LJJ) shows root-mean-squares deviation of 0.6 Å, indicating that the structures that were crystallized under different conditions are virtually identical, and confirms that ordering of the loop 408–426 and small rearrangement of other surrounding loops result from the substrate binding to the active site.

Finally, we observed that the methylated or non-methylated D-galactose residue located at the non-reducing end of the trisaccharide was cleaved by the BpGH2C β-D-galactosidase (Fig 3). Altogether, the enzymology and crystallography experiments conducted in this work revealed the pathway leading to the degradation of the methylated fraction of porphyran. Our observations combined with previous investigations demonstrate that all the methylated and non-methylated fractions of porphyran are digested by the enzymes encoded by porphyran PUL of B. plebeius. Therefore, this PUL can catabolize autonomously the chemically complex porphyran, without the help of enzyme(s) external to this PUL.

Phylogenetic distribution of the porphyran utilizing loci and gene organization

B. plebeius DSM 17135 was the first gut bacteria shown to carry the porphyran PUL. Recently, some strains of B. fragilis, B. ovatus, B. thetaiotaomicron and B. xylanisolvens were further shown to be able to grow on porphyran and to carry a > 96% identical sequence to the B. plebeius porphyran PUL [13]. In order to investigate in more depth whether other human gut bacteria also carry the porphyran PUL, we used BLASTn to identify homologs of the entire coding sequences of PUL-PorA, PUL-PorB and PUL-PorC of B. plebeius DSM 17135 among the 10,969 genomes of bacteria isolated from the human gut and catalogued by Almeida and co-workers [34]. We found that the PULs were present in the genomes of 22 Phocaeicola/Bacteroides strains (corresponding to at least 8 different identified species, S2 Table), including five new species not described so far in Pudlo et al [13]: B. dorei, B. eggerthi, B. stercoris, B. uniformis and B. vulgatus. All these strains came from isolates from the gut microbiome of East Asian individuals, and the same species/strains isolated from other human populations worldwide had no homologs of porphyran PULs.

While the PUL-PorB organization as observed in B. plebieus DSM 17135 was conserved in all the other strains we have identified, the gene organization of PUL-PorA was only conserved in 17/22 strains (Fig 4). In five other strains (B. plebeius strain AM09-36, B. stercoris strain AF05-4, B. uniformis AF21-53, B. uniformis AF26-10BH and B. uniformis strain AM43-9), the homolog of the B. plebieus β-agarase (GH86, Bacple_01694) appeared to have recombined with a DNA fragment composed of two genes predicted to encode metabolic enzymes: 2-dehydro-3-deoxygluconokinase and 2-dehydro-3-deoxyphosphogluconate aldolase (S5 Fig). For these last four strains, a deletion of the genes encoding the GH50 (Bacple_01683) and GH105|GH154 (Bacple_01684) located in PUL-PorC was also observed, highlighting the remodeling of the PUL by insertion and deletion of DNA segments (S5 Fig). Note that parts of PUL-PorA and PUL-PorC were incompletely sequenced in B. plebeius strain AM09-36 and B. eggerthii strain AM42-16.

To explore further the phylogenetic diversity of PUL-PorB, we scanned the whole catalog of Almeida et al [34], including not only the previously mentioned 10,969 gut bacterial isolates, but also 278,263 gut bacterial metagenomes-assembled genomes (MAGs) coming from 13,587 worldwide individuals, which we completed with 29,082 MAGs coming from 842 individuals from under-represented Asian countries (Japan, India, Korea) [35]. We used BLASTn to identify homologs of the six known genes from PUL-PorB and found overall 245 strains having a full PUL-PorB sequence, which were reduced to 130 non-redundant ones after removing strains from the same bacterial species having 100% identity (S3 Table). This analysis revealed eight additional Phocaeicola/Bacteroides species that acquired the porphyran PUL, seven of which had never been described before (B. caccae, B. cellulosilyticus, B. coprocola, B. finegoldii, B. ilei, B. massiliensis and B. stercorirosoris). Interestingly, PUL-PorB was absent in other common Bacteroides sister species from the human gut such as B. intestinalis. We also identified it in two species of Parabacteroides (P. johnsonii and P. merdae), as well as in two new genera: Tyzzerella sp. and Holdemanella sp. which belong to the Bacillota phylum. Overall, the porphyran PUL is thus now known to be present in 19 Bacteroides or Parabacteroides species, and three genera from the Bacillota phylum (Faecalicatena contorta, identified in [13], Tyzzerella and Holdemanella identified here).

In terms of the prevalence of these different PUL-PorB-carrying bacterial species, we observed that four species were the most frequent, with quite equal contributions (B. plebeius, B. uniformis, B. vulgatus, and B. dorei, corresponding each to 15–19% of all positive strains), while others were more anecdotal (<3%), outside of B. coprocola being intermediate (7%) (S6A Fig). Of note, outside of B. plebeius, none of the most frequent species had been identified before.

Genetic diversity of PUL-PorB

The neighbor-joining tree of these 130 non-redundant strains, calculated with the six concatenated protein sequences of PUL-PorB (Fig 5A), revealed four clades with high bootstrap values, allowing us to classify the strains in four groups: GI, GII, GIII and GIIIrec. The percentage of identity of each PUL-PorB protein was compared with the corresponding sequence of B. plebeius DSM 17135 (S3 Table). In the group GI, which includes the B. plebeius DSM 17135 reference, most protein sequences presented 99.5−100% identity with the reference. Similarly, for the members of the group GII, the protein sequences were 99−100% identical to each other, but presented 96−100% identity with the B. plebeius reference. At the root of the GI clade (Outgroup GI), the concatenated sequences of PUL-PorB of 5 strains presented lower identity (98−100%), reflecting some recombination events. For example, the isolated B. plebeius strain AM49-7BH suggested that its divergence with GI members resulted from a recombination event that occurred between SusD, SusC and GH2 genes from GI and Sulf and GH29 genes more related to GII. The recombination site was identified in the gene coding for GH16 (S7 Fig).

The clades GIII encompasses members which presented 92–98% identity with the B. plebeius reference DSM 17135. The PUL-PorB sequence of the GIIIrec members seems to result from a recombination event between GI and GIII (S3 Table), since the SusD (bacple_01704), SusC (bacple_01705) and GH2 (bacple_01706) sequences presented 99–100% identity with GI, while the S1_11 sulfatase (bacple_01701) and GH29 (bacple_01702) were nearly identical to GIII sequences. Again, the recombination site was identified in the gene coding for GH16 (S7 Fig).

Investigating whether there is any correlation between the taxonomic identity of the bacterial species carrying the PUL-PorB and these clades of PUL-PorB, we observed that GI is mostly carried by B. uniformis and B. dorei, while GII is mostly carried by B. vulgatus, B. dorei and B. uniformis, and GIII/GIIIrec is predominantly carried by B. plebeius and B. coprocola (S6B Fig). It thus appears that the three different PUL-PorB groups are not carried by the same set of bacterial species (chi-squared test, p-val < 2.2e-16). Of note, Bacillota are present in the three groups but represent a very minor proportion of bacteria carrying the PUL-porB.

Geographic distribution of PUL-PorB in assembled metagenomes

We then explored the worldwide geographic prevalence of the porphyran degradation system in diverse human gut metagenomes using the catalog from Almeida et al [34] combined with the dataset from Kim et al [35] including 842 individuals from under-represented Asian countries (Japan, India and Korea). This corresponds in total to 14,429 individuals from 32 countries (13 from Europe, 9 from Asia, 6 from America, 2 from Oceania and 2 from Africa). We found that 26% of individuals were positive both in Japan and Korea, while only 3% of individuals were positive in China. All other countries had no hits or prevalence lower than 1% (from higher to lower prevalence: 1/110 positive individual in Mongolia, 1/238 in Fiji, 2/759 in Sweden, 7/3036 in USA, and 2/954 in Israel), as also shown in [13], even though they did not provide quantitative estimates.

Investigating further the prevalence across China by manually downloading and scanning individual metagenomic projects (S4 Table), given that the catalog from Almeida et al [34] only provides a geographic resolution by country, we realized that the prevalence obtained in the original Chinese projects were much higher than in Almeida et al (in average 36% versus 3% [34]). This seems to be because the catalog from Almeida et al [34] applied very stringent filters. Indeed, to have a high-quality catalog, they removed the contigs that had less than 50% completeness and a quality score less than 50. As a consequence, the number of metagenomes per individual are about ten times lower than in the original projects. The dataset compiled by Almeida et al [34] is thus useful to identify which populations carry or not the porphyran PUL, but is not suited to obtain accurate prevalence. We thus manually scanned a total of 20 downloaded projects covering 3,556 biosamples from 10 countries for which gut metagenomes were obtained and assembled (S4 Table). We found that in East Asia, 29% of individuals were positive (585 out of the 2,016 tested) – corresponding to 33% of the Chinese individuals, 25% of the Japanese individuals and 26% of the Korean individuals, while we found only six positive individuals among the 1237 North American gut metagenomes (0.5% of the individuals) and none among the other geographic areas, including 117 individuals from South Asia (India, Bangladesh), 36 from South America (Peru), 139 from Africa (Tanzania, Madagascar) and 11 from Europe (Italy) (S3 Table).

Overall, consistently with previous investigations, the occurrence of algal polysaccharides degradation systems seems to be mostly restricted to East Asia (Japan, China, Korea) and not be present at appreciable frequencies in other Asian countries (Kazakhstan, Mongolia, India, Bangladesh, Singapore) or elsewhere in the world.

The 591 positive individuals from these original 20 metagenome projects were then characterized as belonging to GI, GII, GIII and/or GIIIrec based on their PUL-PorB gene sequence (S5 Table). For 95.4% of the 585 positive East Asian individuals, the genes detected were attributed to a single group; however, few individuals (4.6%) presented PUL-PorB genes that belonged to two different groups. The individuals were then grouped by geographic location (Fig 5B). In order to obtain large sets of individuals, we distinguished populations living in North-East China (Hangzhou), South-East China (Shenzen and Hong-Kong), Korea and Japan. In both set of Chinese individuals, the group GI was present in more than half of the population (57% and 55%, respectively) followed by the group GII (33% and 39%, respectively). In contrast, in Japan, GII was the major group, found in twice higher proportion than GI (55% and 28%, respectively). GIII and GIIIrec were minor groups in these populations, with GIII found in higher proportion in Japan (16%) and GIIIrec in higher proportion in China (14%, 4% and 0% in Hangzhou, Shenzen and Hong-Kong, respectively). In Korea, even though much fewer individuals are analyzed (n = 23), the picture seems to be different from both China and Japan, with GIII being the major group (43%) and GI, GII being at similar frequencies (22% and 26%, respectively).

Analysis of short read datasets

The analyses of the assembled metagenomes revealed that 20% to 30% of individuals carry the PUL-PorB in East Asia (Japan, China, Korea), but that it is mostly absent elsewhere (<1%). In addition, 95.2% of the individuals carry only one version – one group – of the PUL-PorB. These observations may however include some bias inherent to the bioinformatics processes involved in the building up of the contigs. Therefore, to analyze the data devoid of such biases, we have selected a 54 nucleotides sequence allowing to probe the different PUL-PorB groups directly on raw sequencing data (short reads). This allowed us to explore additional metagenomic datasets which were not assembled, thus expending the number of sampling sites, as well as the number individuals tested, with now a total of 4,617 individuals (S4 Table).

As observed previously, we confirmed by this complementary approach that PUL-PorB is not found at appreciable frequencies outside East Asian coastal populations, i.e., in America, Africa or other parts of Asia (Mongolia, India, Malaysia), where the prevalence is null to extremely low (3% at most in Mongolia) (S4 Table, Fig 6). The only exception, however, is in Malaysia where the prevalence in Kuala Lumpur was found to be 16% (but 1% in rural Malaysia), likely reflecting migration fluxes from China. Across Asia, the prevalence obtained are quite higher than the ones obtained with the assembled datasets (50% in China, 72% in Japan and 89% in Korea), with also a certain variance across datasets in Japan (15–94%) and in China (20–70%).

Because of the variability in sequencing depths across datasets, we tested whether the prevalence of PUL-PorB could be influenced by coverage (with the idea that more false negatives could be observed in datasets with too little coverage). However, we did not observe a significant effect of coverage on PUL-PorB prevalence (Pearson correlation coefficient r = 0.14, p-val = 0.499).

We further found that there is no effect of age (chi-squared test, p-val = 0.086), even though metadata on age was available only for two Chinese projects (PRJNA422434 and PRJNA356225); we did not find a significant effect of sex either in the Shenzen dataset (PRJNA422434: chi-squared test, p-val = 0.158).

We then estimated the proportion of bacteria carrying PUL-PorB for each positive individual by dividing the number of reads blasting against PUL-PorB by the total number of reads. We found that it varies between 10^-11 to 10^-7 between individuals (with absolute counts being between 1 and 3099), with a median of 10^-9. Interestingly, Chinese have 3–4 times lower abundances of PUL-PorB than Koreans or Japanese (0.7x10^-9 versus 2.6x10^-9 and 3.2x10^-9, respectively, ANOVA p-val < 2.2e-16, S9 Fig).

Because of the variability in sequencing depths across individuals, we then excluded individuals with too few (less than 10) reads to characterize their PUL-PorB genetic diversity. Among 2138 positive individuals, we thus retained the 1171 individuals having at least 10 reads. The histograms presented in S10 Fig show the number of hits for these individuals and their attribution to the GI, GII, GIII and GIIIrec groups. We found that 60% of individuals carried short reads attributed to a single group, and an additional 21% of individuals had a dominant group, defined as having a prevalence of the major group above 80%. Thus, we estimated that overall, 81% of individuals carried only one or a dominant group. Quite notably, more individuals carried a single or dominant group in China and Japan (84%) as compared to Korea (53%), where half of the individuals carried at least two groups, none of which exceeding 80% in frequency. To verify this is not due to the about five times higher coverage in Korean samples, we looked at the 52 Korean individuals with less than 100 reads (corresponding to individuals with a median number of positive reads of 42, thus quite similar to the median in Japanese of 47 and that in Chinese of 29), and we found a similarly low number of individuals with one or a dominant group (39%) in this subset.

Looking at the prevalence of individuals being positive for each group, we confirmed that GI were predominant in Chinese populations (52% of individuals, versus 33% in Koreans and 32% in Japanese), while GII was most common in Japanese populations (62%, versus 53% in Koreans and 46% in Chinese) (Fig 7). Finally, the GIII and GIIIrec groups were more prevalent in Koreans (60%) as compared to Japanese (25%) and Chinese (14%).

We then tested whether there were significant differences in group prevalences across cities and countries. Including only categories with large enough numbers (more than 10 individuals overall), we found that the six populations were overall significantly different in their group prevalence (X-squared = 324.41, p-value < 2.2e-16) (Fig 7). This difference was mostly driven by significant differences between countries (all comparisons between countries: q-value < 1e-04), as well as between Hong-Kong and Hangzhou, and Hong-Kong and Shenzen/Guangzhou (q-value = 0.027 and 0.044, respectively). We then tested separately which group was significantly different in prevalence between populations and found that all were significantly different (proportion test, q-value < 5e-06), but GIII and GIIIrec presented the stronger difference (proportion test, q-value = 1.1e-15).

Purification of porphyran

6 g of dried Porphyra columbina were suspended in 120 ml de distilled H₂O and autoclaved for 30 min at 120°C. After 14 h at room temperature, the suspension was centrifuged at 8000 rpm during 30 min at 4°C. The supernatant was added to 120 ml of pure ethanol (50% v/v EtOH final concentration) and the solution was maintained at 4°C for 2h. After centrifugation (8000 rpm, 30 min, 4°C), the porphyran, present in the supernatant, was precipitated by addition of 130 ml of pure ethanol (67% v/v EtOH final concentration). After centrifugation the porphyran pellet was dissolved in distilled water, and dialysis for 3 days against distilled water using dialysis membrane with a cut-off 1000 Da (pre-wetted Spectra/Por® 6 dialysis tubing – Spectrum labs). The polysaccharide was lyophilized and the purity was verified by ¹H-NMR. The yield of purification was about 15–20% w/w dried algae.

Heterologous expression of Bacteroides plebeius DSM 17135 PUL-PorB enzymes

The genes encoding the predicted glycoside hydrolases (BpGH29, Bacple_01702; BpGH16C, Bacple_01703; BpGH2C, Bacple_01706) and sulfatase (BpS1_11, Bacple_01701) from B. plebeius DSM 17135 were cloned using genomic DNA as template in the pET-28 or pFO4 expression plasmid [41] without their signal peptides identified with SignalP [42]. The expression strains harboring the recombinant expression plasmids were grown in Luria Bertani (LB) medium supplemented with 50 µg/ml kanamycin (pET28a plasmid) or 100 µg/ml ampicillin (pFO4 plasmid) until the OD_600nm reached 0.6 in a shaking incubator working at 180 rpm and 37°C. After the addition of isopropyl-β-D-thiogalactopyranoside (IPTG), the temperature was cooled down at 20°C and maintained overnight.

Cultures (200 mL) were centrifuged at 6000 g for 15 min and the bacterial pellet was suspended in 10 ml of buffer A (20 mM Tris pH 8, 500 mM NaCl, 20 mM Imidazole). The cells were lysed using a cell disrupter (Constant system Ltd). Insoluble fractions were removed by centrifugation at 20000 g during 30 min at 4°C and the supernatant was loaded on a 1 mL HisTrap^TM HP column (GE Healthcare) connected to a NGC chromatography system (BioRad). The proteins were eluted with a imidazole gradient from 20 to 300 mM gradient of imidazole. Pure enzymes were obtained after a size exclusion chromatography using a HiLoad 16/600 Superdex 75 pg column in buffer B (10 mM Tris-HCl pH 8, 50 mM NaCl).