EP2758528A1 - 3s-rhamnose-glucuronyl hydrolase, procédé de fabrication et utilisations - Google Patents
3s-rhamnose-glucuronyl hydrolase, procédé de fabrication et utilisationsInfo
- Publication number
- EP2758528A1 EP2758528A1 EP12773034.9A EP12773034A EP2758528A1 EP 2758528 A1 EP2758528 A1 EP 2758528A1 EP 12773034 A EP12773034 A EP 12773034A EP 2758528 A1 EP2758528 A1 EP 2758528A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- protein
- sequence
- oligosaccharides
- ulvans
- rhamnose
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
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- HNSDLXPSAYFUHK-UHFFFAOYSA-N 1,4-bis(2-ethylhexyl) sulfosuccinate Chemical compound CCCCC(CC)COC(=O)CC(S(O)(=O)=O)C(=O)OCC(CC)CCCC HNSDLXPSAYFUHK-UHFFFAOYSA-N 0.000 description 8
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- 238000006243 chemical reaction Methods 0.000 description 8
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- AEMOLEFTQBMNLQ-HNFCZKTMSA-N L-idopyranuronic acid Chemical compound OC1O[C@@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-HNFCZKTMSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
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- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 description 1
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- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- 101000856237 Homo sapiens Cancer/testis antigen 1 Proteins 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 108090001090 Lectins Proteins 0.000 description 1
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- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 1
- 239000001099 ammonium carbonate Substances 0.000 description 1
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- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
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- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
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- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
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- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 description 1
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- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
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- 241000701447 unidentified baculovirus Species 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y402/00—Carbon-oxygen lyases (4.2)
- C12Y402/02—Carbon-oxygen lyases (4.2) acting on polysaccharides (4.2.2)
- C12Y402/02023—Rhamnogalacturonan endolyase (4.2.2.23)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y402/00—Carbon-oxygen lyases (4.2)
- C12Y402/02—Carbon-oxygen lyases (4.2) acting on polysaccharides (4.2.2)
- C12Y402/02024—Rhamnogalacturonan exolyase (4.2.2.24)
Definitions
- the present invention relates in particular to proteins, to nucleic acid sequences encoding these proteins, to vectors comprising these coding sequences, to a process for producing these proteins, and to a method for hydrolysis of oligosaccharides using these proteins.
- the present invention finds particular applications in the development of natural bio-resources constituted by organisms and microorganisms comprising ulvans, in particular green algae. In particular, it finds applications in the laboratory, for the analysis of these ulvans, as well as in the food industry, in the field of cosmetics and in the field of pharmaceutical drugs and formulations where the products of degradation of ulvans are upgradable.
- references in brackets [] refer to the list of references at the end of the text.
- Ulvans are composed of different disaccharide repeating units constructed with rhamnose units, glucuronic acids, iduronic acids, xyloses and sulphates.
- aldobiuronic acid or ulvanobiuronic acids, or respectively A (A 3 s) and B (B 3 s), the formulas of which are as follows:
- the unit A (A 3 s) is beta-D-1, 4-glucuronic acid (1 ⁇ 4) alpha-L-1,4-rhamnose 3-sulfate.
- the unit B (B 3S ) is alpha-L-1, 4-iduronic acid (1 ⁇ 4) alpha-L-1,4-rhamnose 3-sulfate.
- Uronic acids are sometimes replaced by xylose residues sometimes sulphated to O-2.
- Ulvans have unique physicochemical properties that make them attractive candidates for new agri-food, pharmaceutical, and cosmetic applications. Ulvans are composed of rare sugars such as rhamnose and iduronic acid. Rhamnose is an important component of the surface antigens of many microorganisms recognized specifically by mammalian lectins. It is also used for flavor synthesis. Iduronic acid is used for the synthesis of glycosaminoglycans (heparin). In addition to the monomers, ulvans and oligo-ulvans have interesting biological properties. Indeed, work has shown, for example, that oligo-ulvans have antitumor, antiviral (anti-influenza) and anticoagulant activities.
- the inventors purified and cloned the ulvan lyase gene specifically cleaving the glycosidic linkage by an elimination mechanism between sulphated rhamnose and uronic acids.
- the degradation products were purified and analyzes of their structures revealed that they were systematically terminated at their non-reducing end by a disaccharide unit composed of glucuronyl acid and sulphated rhamnose.
- ulvan-lyase has made it possible to obtain specific olisaccharides.
- the oligosaccharides obtained can be used, for example, in the cosmetic or medical field, etc.
- the discovery of new enzymes for the degradation or modification of ulvans and oligo-ulvans should make it possible to produce new series of oligosaccharides of molecular weight and well calibrated structures thus widening the possibilities of application and valuation of ulvans.
- the present invention is specifically intended to meet this need by providing proteins that very efficiently hydrolyze oligosaccharides.
- the study of the recognition modalities of the enzymes of the present invention carried out by the inventors demonstrates their 3S-rhamnoglucuronyl hydrolase activity.
- the subject of the present invention is a protein of sequence SEQ ID No. 1 of the attached sequence listing, namely the sequence peptide:
- the protein which is the subject of the invention is a 3S-rhamnoglucuronyl hydrolase.
- the protein which is the subject of the invention may further comprise, at their N-terminal end, a signal sequence or an addressing sequence.
- This signal sequence can be one of signal sequences known to those skilled in the art for the protein, when synthesized in a host cell, to be directed to an organelle or a particular area of the host cell. It may be for example a signal sequence found in sites specializing in the prediction of signal peptides for example http://www.cbs.dtu.dk/services/SignalP/ [2] or even http: // bmbpcu36.leeds.ac.uk/prot_analysis/Signal.html [3]. It may be for example the sequence SEQ ID No. 2 of the attached sequence listing, namely the sequence MNKSILLLVTLLSLYSCT (SEQ ID No. 2). This signal sequence can be cleaved after synthesis of the protein or not.
- the inventors have noted that the signal sequences do not interfere and that their cleavages are not necessary for the expression of protein, for example the protein is overexpressed without its signal peptide.
- the present invention also relates to the nucleic acids encoding the protein of the present invention, in particular for the protein of sequence SEQ ID No. 1.
- the nucleic acid of the present invention may be any sequence coding for the peptide of sequence SEQ ID No. 1 in view of the degeneracy of the genetic code. It may be, for example, a nucleic acid comprising or consisting of the sequence SEQ ID No. 3 of the attached sequence listing, namely the sequence nucleic acid:
- the nucleic acid encoding the protein of the present invention may further comprise, at its 5 'end, the sequence SEQ ID No. 4 of the attached sequence listing, namely a sequence nucleic acid.
- the present invention also relates to an isolated nucleic acid as defined above.
- the present invention also relates to a vector comprising a nucleic acid encoding the protein of the present invention, for example a nucleic acid of sequence SEQ ID No. 3 of the attached sequence listing.
- the vector may be one of the vectors known to those skilled in the art to make proteins by genetic recombination. It is generally chosen in particular according to the chosen cellular host.
- nucleic acids of the present invention or the vectors of the present invention are useful in particular for the genetic recombination of proteins of the present invention.
- the present invention also relates to a host cell comprising a nucleic acid sequence according to the invention or a vector according to the invention.
- the nucleic acid when in a host cell, it can be isolated or not.
- the host cell or host cell may be any suitable host for making the ulna lyase of the present invention from the nucleic acids or vectors of the invention. It can be for example E. coli, Pischia pastoris, Saccharomyces cerevisiae, insect cells, for example a system of insect-baculovirus cells (for example SF9 insect cells using a baculovirus expression system), mammals.
- the host cell may also be the microorganism deposited under number 1-4324 at the CNCM in France.
- the present invention thus also relates to a method of making proteins of the present invention comprising culturing a host cell comprising a nucleic acid sequence according to the invention or a vector or the microorganism deposited under the number I-4324 at the CNCM in France according to the invention.
- This culturing is preferably in a culture medium for the growth of the microorganism. It may be for example ZoBell liquid culture medium, as described in the ZoBell document,
- the culture pH is preferably between 7 and 9, preferably pH 8.
- the culture temperature is preferably between 15 and 30 ° C, preferably 25 ° C.
- the culture is preferably carried out with an NaCl concentration of 20 to 30 gL -1 , preferably 25 gL -1 .
- This method of manufacturing the proteins according to the invention by using the microorganism deposited under the number I-4324 at the CNCM in France or any other host cell transformed for a genetic recombination production according to the present invention may further comprise a step protein recovery according to the invention.
- This recovery or isolation step can be carried out by any means known to those skilled in the art. It may be, for example, a technique chosen from electrophoresis, molecular sieving, ultracentrifugation, differential precipitation, for example ammonium sulphate, ultrafiltration, membrane or gel filtration, exchange of ions, hydroxyapatite elution, hydrophobic interaction separation, or any other known means.
- An example of a method of isolating these 3S Rhamnose glycuronyl hydrolase usable for the implementation of the present invention is described below.
- microorganism or any other host cell transformed for recombinant genetic manufacture according to the present invention may also be used directly to degrade oligosaccharates, in their natural environment or in culture.
- oligosaccharates in their natural environment or in culture.
- it may be a discontinuous or continuous system.
- a culture reactor containing a culture medium suitable for the development of the microorganism can be used.
- the subject of the present invention is also a process for the hydrolysis of oligosaccharides comprising a step of contacting the oligosaccharides with a sequence protein of the invention, by for example a protein of sequence SEQ ID No. 1 or with a host cell comprising a vector with a nucleic acid encoding the protein of the invention, for example a nucleic acid of sequence SEQ ID No. 3. under conditions allowing the hydrolysis of oligosaccharides.
- oligosaccharides are understood to mean oligomers formed by an n number of osues, that is to say monosaccharides by alpha or beta glycosidic linkage. It may be for example di, tri, tetra, oligosaccharides derived from the ulvan degradation by ulvan lyase specifically cleaving the glycosidic bond by a mechanism of elimination between sulfated rhamnose and uronic acids. These degradation products may comprise at their non-reducing end a disaccharide unit composed of glucuronyl acid and a sulphated rhamnose at the 3-position.
- It may also be oligosaccharides having at least one glucuronyl acid bound to a sulphated rhamnose.
- oligosaccharides having at least one glucuronyl acid bound to a sulphated rhamnose.
- a glucuronyl acid bound to sulfated rhamnose itself bound to a uronic acid.
- the pH may also be preferably between 7 and 8, preferably equal to 7.7. This is indeed the optimal pH range.
- the (optimum) temperature is preferably between 40 ° C and 45 ° C.
- the optimum ionic strength may be 100 mM NaCl with 100 mM Tris HCl or 200 mM NaCl.
- the invention advantageously makes it possible to mobilize the very large algae resource currently untapped, in particular of green algae.
- the invention also makes it possible to promote the biodegradation of algae, in particular of green algae, to produce original molecules, which are fragments of oligosaccharides, for example also hydrocolloids for cosmetic, food and pharmaceutical applications or pharmaceutical and parapharmaceutical formulations.
- the degradation products of the oligosaccharides as defined above give access to new products which may be food, cosmetic, pharmaceutical and parapharmaceutical active ingredients used in the agri-food, cosmetic, pharmaceutical and parapharmaceutical fields. These new products can also be non-active products but having a neutrality and / or stability that is very interesting for use in each of these areas.
- the use of the proteins of the present invention further provides access to rare oligosaccharides useful as synthons in glycochemistry.
- the degradation of oligosaccharides can give access to iduronic acid (rare sugar) used for the synthesis of synthetic glycoaminoglycans.
- the present invention also opens new perspectives of use of these algae for applications in bioenergy and chemistry.
- the production of oligosaccharide fragments can give basic molecules for the manufacture of other molecules.
- FIG. 1 is a diagram of the genomic environment of the 3S-rhamnose-glucuronyl hydrolase gene of Percisivirga ulvanivorans. The dark part represents the gene encoding the protein of the invention.
- FIG. 2 represents the protein sequence (SEQ ID No. 1) of the protein of the present invention with the peptide or signal sequence in bold (SEQ ID No. 2).
- FIG. 3 represents the SDS-PAGE electrophoresis gel of the protein of the invention obtained after overexpression in E. coli and purification on an affinity column. The left column represents the molecular weight markers.
- FIG. 4 represents the results obtained from gel permeation chromatography experiments representing the kinetics of modification of oligo-ulvans by the protein of the invention.
- the x-axis represents the retention time in minutes (min)
- the y-axis represents the refractive index.
- FIG. 5 represents the results obtained from ion exchange chromatography experiments conducted with the major degradation products of ulvan by ulvanane lyase of P. ulvanivorans: (A) A-Rha3S; (B) A-Rha3S-GluA-Rha3S; (C) A-Rha3S-1duA-Rha3S; (D) ⁇ -Rha3S-Xyl-Rha3S with the protein of the present invention.
- the x-axis represents the elution time in minutes (min) and the y-axis represents the microsimetric conductimetry (S).
- Figure 6 shows enzymatic reaction patterns catalyzed by the protein of the present invention. After cleavage of the glycosidic bond, the unsaturated monosaccharide produced rearranges spontaneously to 4-deoxy-1-threo-5-hexosulose-uronic acid.
- Figure 7 shows cut-off rate results of glycosidic bond between glucuronyl residue and sulfated rhamnose as a function of oligo-ulvans structure.
- the x-axis represents the time in minutes (min) and the y-axis represents the absorbance (265 nm).
- FIG. 8 is a diagram showing the subsite organization of the active site of the enzyme of the present invention deduced from the experiments presented in FIG. 7.
- the + signs indicate the presence of cationic amino acid potentials necessary for the recognition of the
- FIG. 9 represents the results of proton NMR spectra of the trisaccharides obtained after incubation of oligo-ulvans tetrasaccharides.
- a 10934 base pair DNA fragment was sequenced revealing a group of 6 genes whose corresponding proteins have homologies with families of glycoside hydrolase classified in CAZY (http://www.cazy.org/) and a protein unknown function.
- the primary reactions of TAIL PCR were carried out in 20 ⁇ reactions with 15 ng of genomic DNA, 1 ⁇ GoTaq PCR buffer, 1.5 mM MgCl 2 , 0.2 mM of each dNTP, 0.2 ⁇ l of the first specific primer and degenerate arbitrary primer (5 ⁇ AD1 and AD2, 4 ⁇ AD3, 2 ⁇ AD4 and 3 ⁇ AD5), and 1.25 GoTaq U (Promega).
- the conditions for TAIL PCR side reactions were identical to those for the primary reactions except that 1 ⁇ of a 1:50 dilution of the primary TAIL PCR reaction was used as the original strand and a second specific primer was then used.
- For the tertiary reaction of TAIL PCR 1 ⁇ l of a 1: 50 dilution of the TAIL PCR side reaction was used with the third specific primer.
- the amplification programs were adapted to each of the different TAIL PCR reactions according to the thermocyclers available in the laboratory (Table 2). New GH105 protein gene-specific primers derived from TAIL-PCR experiments were then synthesized to further sequence the gene.
- Figure 1 shows the genomic environment of the ulvan-lyase gene.
- the gene encoding a protein belonging to the GH105 family is 1130 base pairs and the translation leads to a 43.7 kD protein composed of 377 amino acids as shown in FIG. sequence by the SignalP program (http://www.cbs.dtu.dk/services/SignalP/) predicted a 16 amino acid signal peptide with a cleavage site between a serine (S16) and a cysteine (C17 ).
- the complete gene without the signal peptide was cloned and introduced into the pFO4 expression vector according to the following protocol: Primers that match the ends of the GH105 protein gene (excluding its signal peptide) and also possess BamHI restriction and EcoRI respectively at the 5 'and 3' ends of the gene were synthesized. These primers made it possible to amplify the gene according to standard PCR conditions with an annealing temperature of 50 ° C. and 30 cycles.
- PCR products were purified, digested with the appropriate restriction enzymes (BamHI / EcoRI) and ligated into the pFO4 expression vector (modified pET15 (Novagen), Groisillier et al., 2010 Groisillier A, Hervé C, Jeudy A, Rebuffet E, Pluchon PF, Chevolot Y, Flament D, Geslin C, Morgado IM, Power D, Branno M, Moreau H, Michel G, Boyen C, Czjzek M. 2010. MARINE-EXPRESS: taking advantage of high throughput cloning and expression strategies for the post-genomic analysis of marine organisms, Microb Cell Fact 9, 45).
- Luria-Bertani-based expression medium 10 g tryptone, 5 g extract yeast and 10 g NaCl per L
- IPTG isopropyl ⁇ -D1-thiogalactopyranoside
- the bacteria were recovered by centrifugation.
- the pellet of bacteria was suspended in 20 mM Tris-HCl buffer, 500 mM NaCl and 5 mM imidazole pH 7.4.
- the cells were lysed using a French press in a 20 mM Tris-HCl buffer, 20 mM imidazole, 0.5M NaCl and pH 7.4.
- Cell debris was removed by centrifugation.
- the supernatant was injected onto a column of Ni Sepharose loaded with 100 mM NiSO 4 (GE Healthcare). After washing, the retained proteins were eluted with a linear imidazole gradient of 20 mM to 500 mM.
- the active fractions were collected and purified on a superdex 75 column (1.
- Figure 3 shows the gel obtained after migration.
- the protein has been strongly expressed as demonstrated by the electrophoresis gel. Indeed, the most intense band corresponds to a protein whose molecular mass of 42kD corresponds to that expected.
- Enzymes described in the literature belonging to the GH105 family are known to cut the binding between galacturonyl acid and neutral rhamnose of oligosaccharides produced after degradation of rhamnogalacturonan by rhamnogalacturonan lyases.
- Galactose is the C4 epimer of glucose, therefore the formation of the double bond between C4 and C5 of galactose and glucose leads to a uronic acid of the same chemical structure: galacturonyl acid is synonymous with acid glucuronyl.
- Oligosaccharide production was carried out by degradation of polygalacturonan at 1 g / L in 100 mM Tris-HCl at pH 7.7 at 30 ° C with rhamnogalacturonan lyase (Novozymes) at a final concentration of 0.2 ⁇ g / ml The degradation was followed by increasing the absorbance at 235 nm.
- a production of oligo-ulvan was carried out by degradation of ulvan by the ulvan lyase from "01-PN-2010" to the completion followed by an ultrafiltration step on 5000 kD membrane in order to eliminate the resistant fraction.
- the resulting mixture had a concentration of 12.5 mM oligosaccharides, mostly di- and tetra-saccharides but the presence of hexa-, octa- and deca-saccharides was also observed.
- the oligo-ulvans mixture obtained after degradation of ulvane by "01-PN-2010" ulvan-lyase was incubated with the protein of the invention at a final concentration of 0.05 g / ml. room temperature (20 ° C).
- oligo-ulvans The degradation kinetics of oligo-ulvans was followed by gel permeation on a Superdex 200 column coupled in series with a peptide HR column (GE Healthcare). Elution was carried out in 50 mM ammonium carbonate at a flow rate of 0.5 mL min- 1 on an Ultimate 3000 HPLC system (Dionex) equipped with a UV detector (Dionex) at 235 nm and an RI refractometer ( Wyatt) The injected volume was 100 ⁇ .
- the absorbance at 235 nm decreases indicating that the glucuronyl acid of the non-reducing end is removed.
- the peaks detected by their refractive index decreased and then disappeared in favor of new signals as shown.
- HPAEC high-performance ion exchange chromatography
- Dionex ICS 3000 chromatography apparatus equipped with a 20 ⁇ injection loupe, d an automated injection system AS100XR (Thermo Separation Products) and an ion exchange column AS1 1 (4 mm ⁇ 250 mm, Dionex lonPac) associated with a pre-column guard AG1 1 (4 mm ⁇ 50 mm), Dionex lonPac).
- AS100XR Thermo Separation Products
- AS1 1 4 mm ⁇ 250 mm, Dionex lonPac
- pre-column guard AG1 1 (4 mm ⁇ 50 mm
- Dionex lonPac Dionex lonPac
- the mobile phases were ultrapure water (solution A) and 290 mM NaOH (solution B). Elution was carried out at a flow rate of 0.5 mL min -1 and the gradient used was 0 min: 3% Sol.B, 1.5 min: 1% Sol.B, 4.1 min: 5% Sol B: 6.5min: 10% B: 10.0 min: 18% B: 26 min: 22% B: 28 min, 40% B: 30 min: 100% B: 30 , 1 min: 3% B Sol, B: 37 min: 3% Sol B. Data acquisition and analysis were performed using Chromeleon-peak Net software (Dionex).
- the four major oligo-ulvans were purified and then incubated with the protein of the invention.
- the reaction medium was composed of 100 mM Tris HCl pH 7.7, 100 mM NaCl with 125 ⁇ l of purified oligosaccharides and a final protein concentration of 0.05 g / ml.
- the reaction was carried out at 30 ° C and followed with a spectrophotometer at 235 nm. The molecular weight of these four oligosaccharides decreased and the molecules lost their UV absorbing properties at 265 nm.
- Oligosaccharides obtained after incubation of the protein of the invention were analyzed by 1 H proton NMR and carbon. Spectra were obtained with a Bruker Avance DRX 500 spectrophotometer (NMR service from the University of Western Brittany) at 20 ° C. Prior to analysis, the samples were transferred to D 2 O (99.97 atom% D 2 O). The spectra clearly indicate the disappearance of the glucuronyl acid unit and that the oligosaccharides are terminated at their non-reducing end by sulphated rhamnose.
- the structure of the oligosaccharides demonstrate that the protein of the invention is a 3S-rhamnose glucuronyl hydrolase which catalyzes the hydrolysis of the glycosidic bond between glucuronyl acid and sulfated rhamnose. This enzymes catalyze the reactions shown in Figure 6.
- the study of the kinetics of degradation of the oligosaccharides purified by the protein of the invention was carried out in a reaction medium composed of 100 mM NaCl, 100 mM, Tris HCl (pH 7.7) and oligosaccharides of ulvan (dp 2 -8) at 30 ° C in a 1 mL quartz cuvette.
- the maximum concentration of oligo-ulvans used corresponds to an absorbance of 0.5 to 235 nm. 10 ⁇ l of pure GH105 (19 ⁇ g / ml) was added to the reaction medium.
- the degradation of ulvan oligosaccharides (or rather the disappearance of non-reducing glucuronyl acid) was followed by the decrease in absorbance at 235 nm for 5 min.
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Abstract
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FR1158307A FR2980210B1 (fr) | 2011-09-19 | 2011-09-19 | 3s-rhamnose-glucuronyl hydrolase, procede de fabrication et utilisations |
PCT/FR2012/052054 WO2013079828A1 (fr) | 2011-09-19 | 2012-09-14 | 3s-rhamnose-glucuronyl hydrolase, procédé de fabrication et utilisations |
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EP (1) | EP2758528A1 (fr) |
FR (1) | FR2980210B1 (fr) |
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FR3055903B1 (fr) * | 2016-09-13 | 2020-10-16 | Centre Nat Rech Scient | Nouvelle ulvane lyase et son utilisation pour cliver des polysaccharides |
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- 2012-09-14 EP EP12773034.9A patent/EP2758528A1/fr not_active Withdrawn
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IL231595A0 (en) | 2014-05-28 |
FR2980210B1 (fr) | 2014-12-26 |
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