NOVEL ULVAN LYASE AND USE THEREOF FOR CLEAVING POLYSACCHARIDES

Abstract

Some embodiments are directed to a novel ulvan lyase, a nucleic acid sequence coding for said enzyme, a vector comprising said coding sequence, a method for manufacturing said ulvan lyase, and a method for producing ulvan oligosaccharides with biological activity using said enzyme

Claims

1. An ulvan lyase, comprising: a sequence SEQ ID No: 2, 4 or 5, or a sequence having at least 53% sequence identity with the sequence SEQ ID No: 2 or 4; and at its N-terminal end, a signal sequence of sequence SEQ ID No: 6.

2. A nucleic acid coding for the ulvan lyase as defined in claim 1.

3. The nucleic acid as claimed in claim 2, further comprising a sequence SEQ ID No: 1 or 3.

4. A vector, comprising: the nucleic acid as claimed in claim 2.

5. A host cell, comprising: the nucleic acid sequence as claimed in claim 2.

6. A process for production of the ulvan lyase as defined in claim 1 by genetic recombination utilizing a nucleic acid coding for the ulvan lyase.

7. A process for degradation of ulvans, comprising: a stage of contacting ulvans with the ulvan lyase as claimed in claim 1, under conditions enabling the degradation of the ulvans by enzymatic digestion by the ulvan.

8. A method of using the ulvan lyase as defined in claim 1 for the production of oligo-ulvans.

9. A vector, comprising: the nucleic acid as claimed in claim 3.

10. A host cell, comprising: the nucleic acid sequence as claimed in claim 3.

11. A host cell, comprising: the vector as claimed in claim 4.

12. A process for production of the ulvan lyase of claim 1 by genetic recombination utilizing a nucleic acid coding for a ulvan lyase that includes a sequence SEQ ID No: 1 or 3.

13. A process for production of the ulvan lyase of claim 1 by genetic recombination utilizing a vector that includes a nucleic acid coding for the ulvan lyase.

14. A process for degradation of ulvans, comprising: a stage of contacting ulvans with the host cell as claimed in claim 5, under conditions enabling the degradation of the ulvans by enzymatic digestion by the host cell.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0032] FIG. 1 shows the comparison of the expression in E. coli BI2(DE3) of the catalytic module of the ulvan lyase of N. ulvanivorans PLR (A and B) and F. agariphila DSM 15362 (C and D). Plates A and C correspond to SDS-PAGE analyses. Plates B and D correspond to Western blot analyses with an anti-His-Tag antibody. FW=Flow-through of the nickel affinity chromatography; E1 and E2: eluates 1 and 2 of the nickel affinity chromatography.

[0033] FIG. 2 shows an activity test of the recombinant ulvan lyase BN863_22190_cat as a function of the NaCl concentration (A), of the nature of the buffer (B) and of the pH (for the best buffer, C). The activity was followed by spectrophotometry at 235 nm.

[0034] FIG. 3 shows a FACE analysis (Fluorophore-assisted Carbohydrate Electrophoresis) of the oligo-ulvans liberated by extensive digestion of ulvan by the recombinant ulvan lyase BN863_22190_cat. These samples are named FracX where X is a fraction number corresponding to the purification of these oligosaccharides by size exclusion chromatography. The oligosaccharides DP2 and DP4 are standard oligo-ulvans liberated by the wild ulvan lyase of Nonlabens ulvanivorans PLR [1].

[0035] FIG. 4 shows a mass spectrum of the fraction 48 recorded on the BIBS platform (INRA, Nantes). The mass/charge (m/z) main peaks correspond to a disaccharide -R3S.

[0036] FIG. 5 represents a mass spectrum of the fraction 39 recorded on the BIBS platform (INRA, Nantes). The mass/charge (m/z) main peaks correspond to a tetrasaccharide -R3S-Xyl-R3S.

EXAMPLES

Example 1: Expression of the Ulvan Lyase of Formosa agariphila

[0037] The gene coding for that ulvan lyase (BN863_22190) was identified in the genome of F. agariphila DSM 15362 by sequence homology with the ulvan lyase of Nonlabens ulvanivorans PLR (formerly Persicivirga ulvanivorans) [2, 3].

[0038] The protein encoded by this gene has a molecular mass of 56,630 Da (516 residues). It has a modular architecture with an N-terminal signal peptide followed by a catalytic module, a module of unknown function and a C-terminal type IX secretion module. Over the totality of its sequence, this protein has 52% sequence identity with that of the ulvan lyase of N. ulvanivorans PLR, as determined by sequence alignment by the CLUSTALW software. The catalytic module of the ulvan lyase of F. agariphila DSM 15362 (residues 25-285, 28,738 Da) exhibits 66% sequence identity with the sequence of the catalytic module of the ulvan lyase of N. ulvanivorans (residues 24-291).

[0039] The nucleotide sequence corresponding to the catalytic module of the ulvan lyase of F. agariphila DSM 15362 was cloned into the vector pFO4 (derived from the commercial vector pET15b, [6]), and the corresponding recombinant protein was overexpressed in Escherichia coli (SEQ ID No: 5). For comparison, a similar study was performed for the catalytic module of the ulvan lyase of N. ulvanivorans PLR.

[0040] The results presented in FIG. 1 show that the catalytic module of the ulvan lyase of N. ulvanivorans PLR is not visible by electrophoreses on denaturing polyacrylamide gel stained with Coomassie blue (FIG. 1A), even after purification by nickel affinity chromatography. This enzyme was nonetheless weakly detected by an anti-Histidine-TAG antibody (FIG. 1B, see arrow); which confirmed very weak soluble expression of this protein (a few g per liter of culture). In contrast, the catalytic module of the ulvan lyase of F. agariphila DSM 15362 (referred to below as BN863_22190_cat) is very strongly expressed in soluble form, as indicated by the wide band visible at 29 kDa in the SDS-PAGE (FIG. 1C). This was confirmed by Western blot (FIG. 1D), which is a much more sensitive technique than SDS-PAGE. The production yield of this recombinant protein was 30 mg per liter of culture, that is to say at least 10,000 times more than the recombinant ulvan lyase of N. ulvanivorans PLR.

Example 2: Biological Activity of the Ulvan Lyase of Formosa agariphila

[0041] An activity test typically used for the polysaccharide lyases (e.g. [7, 8]) was performed for BN863_22190_cat. It can include in following the absorbance of the reaction medium at 235 nm. In fact, the polysaccharide lyases liberate oligosaccharides exhibiting an unsaturated monosaccharide at the non-reducing end which absorb at 235 nm. This test thus made it possible to determine the optimal salinity of the medium for the enzyme (FIG. 2A), the optimal buffer (FIG. 2B) and the optimal pH with the better buffer (FIG. 2C).

[0042] An extensive degradation of a pure ulvan (ULV100, Elicityl, France) was also performed utilizing BN863_22190_cat. The oligo-ulvans liberated were purified by size exclusion chromatography on a system of three interconnected Superdex 30 columns (GE Healthcare, France). These purified oligosaccharides were then analyzed by the technique called FACE (Fluorophore-assisted Carbohydrate Electrophoresis). The reducing end of the oligosaccharides was thus labeled with a fluorescent molecule (8-amino-naphthalene-1,3,6-trisulfonic acid, ANTS) as previously described [9]. The labeled oligosaccharides are then analyzed by polyacrylamide gel electrophoresis. Oligo-ulvans previously obtained in the laboratory by the wild ulvan lyase of N. ulvanivorans PLR [2] were also utilized as standards (DP2: the disaccharide -R3S; DP4: the tetrasaccharide -R3S-Xyl-R3S; the structures of DP2 and DP4 have been determined by NMR [2]). FIG. 3 shows that the terminal oligosaccharides Fraction 48 and Fraction 39 migrate at the same size as the standards DP2 and DP4, respectively. The structure of these oligosaccharides generated by BN863_22190_cat. was confirmed by mass spectrometry (BIBS platform, INRA, Nantes) (FIGS. 4 and 5). Consequently, like the wild ulvan lyase of N. ulvanivorans PLR, BN863_22190_cat. cleaves the beta-1,4 bond between the D-glucuronic acid (GlcA)/L-iduronic acid (IduA) residues and the rhamnose-3-sulfate (R3S) endolytically liberating oligosaccharides of different sizes exhibiting an unsaturated sugar A at the non-reducing end. The main end products are the disaccharide -R3S, and the tetrasaccharide -R3S-Xyl-R3. The tetrasaccharides -R3S-GlcA-R3S and A-R3S-IduA-R3S also described in the article Nyvall Collen et al., JBC, 2011 are not observed in the experiment owing to the extensive degradation of the ulvan by BN863_22190_cat. In fact, these two types of tetrasaccharide are cleaved in two by BN863_22190_cat, liberating -R3S disaccharides (FIG. 3). In contrast, the tetrasaccharide -R3S-Xyl-R3S is an end product resistant to the enzymatic cleavage (FIG. 3), owing to the presence of a xylose residue at position 3 from the non-reducing end.

[0043] The results show a strong ulvan degradation activity by BN863_22190-cat, confirmed by measurement of the absorbance at 235 nm and by the electrophoresis analysis and by mass spectrometry of the purified oligo-ulvans. By comparison, a residual activity was detected on following the degradation of the ulvan by the ulvan lyase of N. ulvanivorans by spectrophotometry at 240 nm (data not shown).

REFERENCES

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