Enzymatic method for removing sulphite

Abstract

The present invention relates to an enzymatic method for removing sulphite. In particular, the present invention relates to a method for converting sulphite present in a composition into thiosulphate, comprising the introduction of 3-mercaptopyruvate sulphurtransferase (3-MST) and a substrate of said 3-mercaptopyruvate sulphurtransferase into said composition. The present invention can be used in particular in the pharmaceutical, cosmetic, food and chemical fields, and any field involving the use of sulphite.

Claims

1. A method for treating a composition, comprising introducing into said composition 3-mercaptopyruvate sulfurtransferase (3-MST) and a substrate of said 3-mercaptopyruvate sulfurtransferase in order to convert sulfites in the composition into thiosulfates, wherein the composition is an agri-food, pharmaceutical, cosmetic or veterinary composition.

2. A method for converting sulfite present in a composition into thiosulfate, comprising introducing into said composition 3-mercaptopyruvate sulfurtransferase (3-MST) and of a substrate of said 3-mercaptopyruvate sulfurtransferase and converting the sulfite present in the composition into thiosulfate, wherein the composition is an agri-food, pharmaceutical, cosmetic or veterinary composition.

3. The method as claimed in claim 1, wherein said 3-mercaptopyruvate sulfurtransferase is recombinant.

4. The method as claimed in claim 1, wherein said 3-mercaptopyruvate sulfurtransferase comprises, in its peptide sequence, the following three sequences: TABLE-US-00004 (SEQ ID No. 13) RX.sub.aWWM (SEQ ID No. 14) CGSGVTAX.sub.b (SEQ ID No. 15) GHIX.sub.cG wherein X.sub.a is A, V or L, X.sub.b is A or C, and X.sub.c is P or E.

5. The method as claimed in claim 1, wherein said 3-mercaptopyruvate sulfurtransferase is from Escherichia coli.

6. The method as claimed in claim 1, wherein the 3-mercaptopyruvate sulfurtransferase is a protein or peptide sequence selected from the group SEQ ID Nos 2, 5, 6 and 7.

7. The method as claimed in claim 1, wherein said substrate of said 3-mercaptopyruvate sulfurtransferase is 3-mercaptopyruvate.

8. The method as claimed in claim 1, wherein the stoichiometric ratio of said substrate to the sulfite present in said composition is at most 1:1.

9. The method as claimed in claim 2, wherein said 3-mercaptopyruvate sulfurtransferase is recombinant.

10. The method as claimed in claim 2, wherein said 3-mercaptopyruvate sulfurtransferase comprises, in its peptide sequence, the following three sequences: TABLE-US-00005 (SEQ ID No. 13) RX.sub.aWWM (SEQ ID No. 14) CGSGVTAX.sub.b (SEQ ID No. 15) GHIX.sub.cG wherein X.sub.a is A, V or L, X.sub.b is A or C, and X.sub.c is P or E.

11. The method as claimed in claim 2, wherein said 3-mercaptopyruvate sulfurtransferase is from Escherichia coli.

12. The method as claimed in claim 2, wherein the 3-mercaptopyruvate sulfurtransferase is a protein or peptide sequence selected from the group SEQ ID Nos 2, 5, 6 and 7.

13. The method as claimed in claim 2, wherein the composition is an agri-food, pharmaceutical, cosmetic or veterinary composition.

14. The method as claimed in claim 2, wherein said substrate of said 3-mercaptopyruvate sulfurtransferase is 3-mercaptopyruvate.

15. The method as claimed in claim 2, wherein the stoichiometric ratio of said substrate to the sulfite present in said composition is at most 1:1.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 is a scheme representing the catalytic mechanism of 3-mercaptopyruvate sulfurtransferase in which 3-mercaptopyruvate is used as sulfur-donating substrate and sulfite is used as acceptor resulting in the formation of pyruvate and of thiosulfate.

(2) FIG. 2 is a photograph of an SDS-12.5% PAGE protein gel showing the various steps of purification of Escherichia coli 3-mercaptopyruvate sulfurtransferase. The MW column corresponds to the molecular weight markers, the SSO column corresponds to the soluble fraction obtained after bacterial cell lysis, the C65% column corresponds to the insoluble fraction obtained after precipitation in the presence of 65% ammonium sulfate, the Ph-Seph column corresponds to the fraction obtained after Phenyl-Sepharose hydrophobic interaction chromatography (GE Healthcare (registered trade mark)) and the Q-Seph column corresponds to the fraction obtained after Q-Sepharose anion exchange chromatography (GE Healthcare (registered trade mark)).

(3) FIG. 3 is a simplified scheme representing the principle of the SO kit comprising the use of two enzymes, each catalyzing a reaction step. The first step involves sulfite oxidase (reaction 1) and the second involves NADH peroxidase (reaction 2).

(4) FIG. 4 represents the monitoring of the absorbance at 340 nm as a function of time of an untreated white wine sample (black curve white wine alone) and of a white wine sample after treatment with 2 μM of 3-mercaptopyruvate sulfurtransferase and a stoichiometric (1:1) amount of 3-mercaptopyruvate relative to the sulfites (upper curve white wine+3-MST+3-MP).

(5) FIG. 5 represents the monitoring of the absorbance at 340 nm as a function of time of a white wine sample after treatment with a stoichiometric (1:1) amount of 3-mercaptopyruvate relative to the sulfites (black curve white wine alone), of a white wine sample after treatment with 2 μM of 3-mercaptopyruvate sulfurtransferase (white wine+3-MST) and of a white wine sample after treatment with 2 μM of 3-mercaptopyruvate sulfurtransferase and a stoichiometric (1:1) amount of 3-mercaptopyruvate relative to the sulfites (upper curve white wine+3-MST+3-MP).

(6) FIG. 6 represents the monitoring of the absorbance at 340 nm as a function of time of an untreated white wine sample (black curve white wine alone), of white wine samples after treatment with 2 μM of 3-mercaptopyruvate sulfurtransferase and a 3-mercaptopyruvate/sulfite ratio equal to 1/4 (curve 1/4 of 3-MP), to 1/2 of (curve 1/2 of 3-MP) and to 3/4 (curve 3/4 of 3-MP).

(7) FIG. 7 is a graph representing the percentage of residual sulfite after treatment with said process (y-axis) as a function of time (x-axis).

(8) FIG. 8 represents the monitoring of the absorbance at 340 nm as a function of time of an untreated red wine sample (black curve red wine alone) and of a red wine sample after treatment with 0.5 μM of 3-mercaptopyruvate sulfurtransferase and a stoichiometric (1:1) amount of 3-mercaptopyruvate relative to the sulfites (upper curve red wine+3-MST+3-MP).

(9) FIG. 9 represents the monitoring of the absorbance at 340 nm as a function of time of a local anesthetic (Septanest (registered trade mark)) after treatment with 0.5 μM of 3-mercaptopyruvate sulfurtransferase and 3-mercaptopyruvate with a 3-mercaptopyruvate/sulfite ratio equal to 0 (curve 0 3-MP/sulfite), to 1/2 (curve 1/2 of 3-MP/sulfite) and to 1 (curve Septanest+3-MST+3-MP).

(10) FIG. 10 represents an alignment of protein sequences and of secondary structures of three 3-mercaptopyruvate sulfurtransferases (3-MST): Escherichia coli 3-MST (code PDB 1URH), human 3-MST (code PDB 4JGT) and Leishmania major 3-MST (code PDB 10KG) demonstrating the conserved amino acids and the position of the amino acid (Aspartate 17) that has been mutated to alanine.

(11) FIG. 11 represents a scheme of the pET20b 3-MST vector used for the production of the wild-type form and of the D17A mutant of the Escherichia coli 3-mercaptopyruvate sulfurtransferase. The pET20b 3-MST construct is a recombinant vector obtained from the commercial pET20b, Invitrogen (registered trade mark).

(12) FIG. 12 corresponds to the sequence of the coding region of the Escherichia coli 3mst gene. The mutated codon which makes it possible to obtain the D17A mutant of Escherichia coli 3-mercaptopyruvate sulfurtransferase is underlined and highlighted in gray.

(13) FIG. 13 is the protein sequence of the wild-type form of Escherichia coli 3-mercaptopyruvate sulfurtransferase. The aspartic acid underlined and highlighted in gray corresponds to the amino acid mutated to alanine in the D17A mutant of Escherichia coli 3-mercaptopyruvate sulfurtransferase.

(14) FIG. 14 contains the sequence of the forward (a) and reverse (b) nucleotide primers used during the site-directed mutagenesis carried out on the pET20b 3-MST expression vector.

EXAMPLES

Example 1: Production and Purification of Escherichia coli 3-Mercaptopyruvate Sulfurtransferase D17A

(15) The plasmid encoding Escherichia coli 3-mercaptopyruvate sulfurtransferase D17A was obtained by site-directed mutagenesis of the plasmid encoding the wild-type form of the enzyme (FIGS. 11, 12, 13 and 14).

(16) The D17A mutant of Escherichia coli 3-mercaptopyruvate sulfurtransferase was produced by transformation of Escherichia coli BL21(DE3) bacteria with a pET20b 3-MST expression vector. The transformation of 50 μl of BL21(DE3) competent cells with 100 ng of pET20b 3-MST was carried out by incubating the mixture in ice for 30 min, followed by a heat shock (30 seconds at 42° C.). The mixture was then used to inoculate a preculture of 50 ml of LB medium supplemented with ampicillin (200 mg/l). After overnight incubation at 37° C. with shaking, 4 l of LB medium supplemented with ampicillin (200 mg/l) were inoculated at 1/100 and placed at 37° C. with shaking. When the optical density at 600 nm reached 0.6, the expression was induced by adding 1 mM of IPTG (isopropyl-1-thio-β-D-galactopyranoside). The cells were then placed at 37° C. for 3 hours with shaking, then harvested by centrifugation at 3000 g. The cell pellets were taken up in 20 ml of TE buffer 50 mM Tris HCl, 2 mM EDTA, pH 8) containing 10 mM of DTT, 20 U/ml of benzonase and 4 mM of MgSO.sub.4. The cell lysis was carried out by sonication at 4° C. at a power of 40 W per period of two times 2 minutes for 50% of the time. The cell debris was removed by centrifugation at 17 000 g for 45 minutes.

(17) The enzyme was purified by means of a protocol comprising the following three steps: Precipitation with ammonium sulfate: The soluble fraction obtained after sonication was brought to 65% ammonium sulfate (AS) saturation with gentle stirring at 4° C. At this concentration, the protein precipitated and, after centrifugation at 17 000 g for 45 minutes, the pellet was then taken up in 20 ml of TE buffer (50 mM Tris HCl, 2 mM EDTA, pH 8). Phenyl-Sepharose hydrophobic interaction chromatography (GE Healthcare (registered trademark)): Ammonium sulfate was gradually added to the protein solution until a concentration of 1 M was obtained. This solution was then filtered (0.2 μm) before injection onto a Phenyl-Sepharose hydrophobic interaction column pre-equilibrated with TE buffer, 1M AS. The elution was carried out by decreasing gradient of ammonium sulfate (from 1 to 0 M). The fractions collected were analyzed by SDS-12.5% PAGE electrophoresis and those containing the 3-mercaptopyruvate sulfurtransferase were combined and then dialyzed overnight against TE buffer (50 mM Tris HCl, 2 mM EDTA, pH 8). Q-Sepharose anion exchange chromatography (GE Healthcare (registered trademark)): After dialysis, the protein solution was filtered at 0.2 μm and then injected onto a Q-Sepharose anion exchange chromatography column pre-equilibrated with TE buffer (GE Healthcare (registered trade mark)). The elution was carried out by applying an increasing gradient of KCl (0 to 1 M). The fractions containing the 3-mercaptopyruvate sulfurtransferase were identified by SDS-12.5% PAGE electrophoresis and then combined and concentrated by ultrafiltration on an Amicon cell using a YM 10 membrane (10 kDa threshold). The protein solution thus purified was brought to an ammonium sulfate saturation of 70% in order to precipitate the protein and to store it at −20° C. in the presence of 5 mM of DTT.

(18) FIG. 2 is a photograph of an SDS-12.5% PAGE protein gel after migration of the samples obtained after each of the Escherichia coli 3-mercaptopyruvate sulfurtransferase purification steps. This figure clearly shows the various purification steps demonstrating the obtaining of the 3-mercaptopyruvate sulfurtransferase purified to homogeneity.

Example 2: Removal of the Sulfite Present in White Wine

(19) The 3-mercaptopyruvate sulfurtransferase used was the D17A mutant of the purified Escherichia coli 3-mercaptopyruvate sulfurtransferase obtained in example 1 above.

(20) The study was carried out with a dry white wine (Colombard Sauvignon, Côte de Gascogne, 44330 La chapelle Heulin, France). The sulfite concentration in this wine was measured at 160 mg/l before treatment using the sulfite oxidase kit (SO kit) (Megazyme International, Ireland) (black curve, FIG. 4). The amount of sulfite is given by the difference in optical density at 340 nm (ΔOD340 nm) measured using a spectrophotometer (SAFAS UVmc.sup.2).

(21) The optical density was measured at 340 nm since it makes it possible to monitor the amount of NADH oxidized to NAD.sup.+ (reaction 2, FIG. 3) which correlates with the amount of sulfite converted into sulfate (reaction 1, FIG. 3). In other words, the decrease in the optical density corresponds to the oxidation of the sulfites by sulfite oxidase, thus demonstrating the presence of sulfite.

(22) The SO kit makes it possible to assay the amount of total sulfites in the wine, that is to say the free sulfites and the sulfites combined with organic molecules. Said method has the advantage of removing the total sulfites; in point of fact, the forms combined once in the stomach at a pH of 1.5 to 5 are released and constitute a not insignificant source of sulfite in the organism that it is necessary to eliminate. If the method makes it possible to remove the total sulfites, that means that the removal of the free sulfite forms results in a shift in the chemical equilibrium toward the decombination of the bound sulfites and thus, in the end, in the total removal of the sulfites (Grignard et al., 1950, Traité de chimie organique [Treatise on organic chemistry [14]).

(23) In order to determine whether the 3-mercaptopyruvate sulfurtransferase effectively makes it possible to convert the sulfite into thiosulfate, 25 ml of white wine were mixed with 2 μM of 3-mercaptopyruvate sulfurtransferase and 3-mercaptopyruvate in stoichiometry of 1:1 relative to the sulfite. The whole mixture was incubated for 5 min at a temperature of 25° C. and the optical density at 340 nm was measured. FIG. 4 presents the results obtained.

(24) The optical density was measured and there is no longer any decrease in the absorbance, attesting to a total removal of the sulfite present in the wine, demonstrating the conversion of the sulfite into thiosulfate (curve white wine+3-MST+3-MP, FIG. 4). Various controls show that the removal of the sulfite is indeed linked to the enzymatic activity of the enzyme, since the same experiment carried out in the absence of the 3-mercaptopyruvate substrate or in the absence of enzyme does not bring about a drop in the amount of sulfite in the wine. The results obtained are represented in FIG. 5 (respectively, curves white wine+3-MST and white wine+3-MP).

(25) The removal of the free sulfites from white wine was also demonstrated by using strips (Quantofix (registered trade mark) Sulfite, Macherey-Nagel) (results not disclosed).

(26) On the other hand, the amount of sulfite after the method could not be determined by the conventional methods currently used in the wine making industry, such as: a. the Franz Paul method since it requires strong and aggressive acidification by adding sulfuric acid and heating, which under these conditions results in the dismutation of the thiosulfate to sulfite, b. the use of chemical kits since they contain reagents specific for thiol groups; in point of fact, the thiosulfate and the enzyme have thiolate groups which constitute a source of “parasitic” reactions that distort the measurement.

(27) The assaying of sulfite after the implementation of an example method in accordance with the present invention was carried out using the SO kit (Megazyme International, Ireland) which constitutes a quantitative specific assaying method, and using strips (Quantofix (registered trade mark) Sulfite, Macherey-Nagel) making it possible to validate this method as a qualitative test.

(28) As mentioned above, the SO kit makes it possible to assay the amount of total sulfites in the wine, that is to say the free sulfites and the sulfites combined with organic molecules. This assaying was carried out according to the recommended protocol in a glass vessel by mixing 1.3 ml of distilled water, 250 μl of buffer, 100 μl of sample studied, 100 μl of NADH and 10 μl of NADH peroxidase. After stabilization of the absorbance, 10 μl of sulfite oxidase were added and the variation in absorbance at 340 nm was measured.

(29) In order to determine the optimal concentration of 3-mercaptopyruvate sulfurtransferase to be used, tests were carried out in the presence of 3-mercaptopyruvate in a stoichiometric amount relative to the sulfites and of variable concentrations of 3-mercaptopyruvate sulfurtransferase. The optical density at 340 nm was measured instantaneously following the reaction mixed.

(30) The concentration of sulfite in the white wine sample was 160 mg/l. A measurement of the residual sulfite concentration after implementation of the method was carried out using the SO kit. The results obtained in the presence of various concentrations of 3-mercaptopyruvate sulfurtransferase are presented in table 1 below and in FIG. 5.

(31) TABLE-US-00002 TABLE 1 Residual amount of sulfite as a function of 3-mercaptopyruvate sulfurtransferase concentration Concentration of enzyme (3-MST) in nM 100 250 350 450 500 Concentration of 160 96 50 25 <10 residual sulfite (mg/l)

(32) As demonstrated above, the 3-mercaptopyruvate sulfurtransferase makes it possible to remove the sulfites present in the white wine and a concentration of 500 nM of enzyme makes it possible to remove all the sulfites present.

(33) Additionally, tests in the presence of variable concentrations of 3-mercaptopyruvate and of 500 nM of 3-mercaptopyruvate sulfurtransferase were carried out on a white wine sample. In particular, 3-mercaptopyruvate/sulfite molar ratios of 1/4, 1/2 and 3/4 were tested. A measurement of the residual sulfite concentration after implementation of the method was carried out using the SO kit. The results obtained in the presence of various concentrations of 3-mercaptopyruvate are presented in table 2 below and in FIG. 6.

(34) TABLE-US-00003 TABLE 2 Residual amount of sulfite as a function of 3-mercaptopyruvate concentration Percentage of 3-mercaptopyruvate/sulfite (mol/mol) 0 25 50 75 95 100 Residual sulfite 168 112 86 46 13.4 <10 concentration (mg/l)

(35) As demonstrated in table 2 above and in FIG. 6, the method according to the invention also advantageously makes it possible to control the final sulfite concentration.

(36) In other words, the method according to the invention advantageously makes it possible to modify the sulfite concentration in a composition/sample to a given value or to eliminate/convert the sulfites present in a composition, for example wine.

(37) Finally, thiosulfate is a chemically stable molecule. A verification of its stability in wine was carried out. For that, the residual amount of sulfite after treatment was measured as a function of time according to the following method: once the removal method had been applied to a volume of 25 ml of wine according to the same protocol as described above (500 nM 3-mercaptopyruvate sulfurtransferase, with a stoichiometric amount of 3-mercaptopyruvate/sulfite), the sample was stored at ambient temperature for 2 weeks and the amount of residual sulfate was quantified at regular time intervals, namely every three days. The results obtained show that, over a period of 17 h (or two weeks, results not shown), there is no release of sulfite by dismutation of the thiosulfate in the wine (FIG. 7).

(38) In other words, a method in accordance with the present invention makes it possible to remove the sulfites by modifying them into thiosulfates in a composition, the modification being stable over time.

Example 3: Removal of the Sulfite in Red Wine

(39) In the present example, the composition was red wine (Corbières, France). Since the tannins present in red wine interfere with the sulfite quantification method (SO kit mentioned above), it was necessary to remove them in order to demonstrate the disappearance of the sulfite in the composition.

(40) The method for removing the sulfites was carried out on red wine according to the following protocol: 500 nM of 3-mercaptopyruvate sulfurtransferase, stoichiometric amount of 3-mercaptopyruvate/sulfite present in this wine. The tannins were removed by precipitation in the presence of polyvinylpolypyrollidone (PVPP) according to the following method: 10 ml of red wine sample were treated with 0.2 g of PVPP and a centrifugation at 4000 g was carried out for 5 minutes. This step was repeated 3 times.

(41) A measurement of the sulfite concentration before and after implementation of the method was carried out using the SO kit according to the same protocol as described in example 1. The results obtained are presented in FIG. 8. The sulfite concentration in the red wine was 100 mg/l (black curve red wine alone). The addition of 500 nM of 3-mercaptopyruvate sulfurtransferase and of a stoichiometric amount of 3-mercaptopyruvate made it possible to remove the sulfite (upper curve red wine+3-MST+3-MP).

(42) This example clearly demonstrates that a method according to the invention makes it possible to remove the sulfites present in red wine.

Example 4: Removal of the Sulfite in an Anesthetic

(43) In this example, the composition was a local anesthetic (Septanest (registered trade mark)) 40 mg/ml, with adrenalin at 1/100 000, articaine Chl., 40 mg/l, Laboratoire Septodont).

(44) The method for removing the sulfites from the composition was carried out according to the following protocol: 500 nM of 3-mercaptopyruvate sulfurtransferase, stoichiometric amount of 3-mercaptopyruvate/sulfite present in the composition.

(45) The sulfite concentration before and after implementation of the method was measured using the SO kit according to the same protocol as described in example 1. The results obtained are presented in FIG. 9. The sulfite concentration in the composition was 410 mg/l (curve 0 3-MP/sulfite). The addition of 500 nM of 3-mercaptopyruvate sulfurtransferase and of a stoichiometric amount of 3-mercaptopyruvate allowed the total removal of the sulfite present in the composition (curve Septanest+3-MST+3-MP) in accordance with the present invention.

(46) In addition, the method was carried out with a 1/2 ratio of 3-mercaptopyruvate/sulfite (mol/mol) (FIG. 9, curve 1/2 of 3-MP/sulfite). As demonstrated, half the sulfites present in the composition are converted into thiosulfate.

(47) This example thus clearly demonstrates that a method in accordance with the present invention makes it possible to treat pharmaceutical compositions in order to reduce the amount of sulfite present.

LIST OF REFERENCES

(48) 1. Shih et al., 1977, The New England Journal of Medicine. 2. Andersson et al., 2013, International Archives of Occupational and Environmental Health. 3. Ozsoy et al., 2014, Toxicology and Industrial Health. 4. WO 2015/051187. 5. Hermann et al., 2015, “The octahaem MccA is a haem c-copper sulfite reductase” Nature 520, 706-709 6. http://www.promega.com/vectors/mammalian_express_vectors.htm. 7. http://www.qiagen.com/overview/qiagenes.aspx?gaw=PROTQIAgenes0807&gkw=mammalian+expression 8. http://www.scbt.com/chap_exp_vectors.php?type=pCruzTM%20Expression%20Vectors. 9. WO 83/004261 10. Beutler, H. O., 1988, Sulphite In Methods of Enzymatic Analysis. 11. Bondet and Sylvestre, 2005, Pratiquer les contrôles en cenologie [Carrying out tests in enology]. 12. FDA report regarding sodium thiosulfate, SCOGS-52, 1975. 13. Arrestier et al., 2016, Medicine, 95 (6). 14. Grignard et al., 1950, Traitè de chimie organique [Treatise on organic chemistry], 769-770.