[0001] The present invention relates to a method for producing L-methionine by enzyme reaction between a precursor of L-methionine, dimethyl disulfide (DMDS) and a reducing organic compound. It also relates to a two-step process for producing L-methionine by enzyme reaction between a precursor of L-methionine and methyl mercaptan, the latter being obtained by enzymatic hydrogenolysis of DMDS.

[0002] Methionine is an essential amino acids the human body and is widely used as an additive for animal feed. It is also used as a raw material for pharmaceuticals. Methionine acts as a precursor of such compounds as choline (lecithin) and creatine. It is also a raw material for synthesis of cysteine ​​and taurine.

[0003] S-adenosyl-L-methionine (SAM) is a derivative of L-methionine and is involved in the synthesis of various neurotransmitters in the brain. L-methionine and / or SAM, inhibit fat accumulation in the body and improves blood circulation in the brain, heart and kidneys. L-methionine can also be used to promote digestion, detoxification and excretion of toxic substances or heavy metals such as lead. It has an anti-inflammatory effect on bone and joint diseases and is also an essential nutrient for the hair, preventing their premature and unwanted fall.

[0004] Methionine is already known to be prepared industrially by chemical pathways from raw materials derived from petrochemicals, as described for example in FR2903690 documents, WO2008006977, US2009318715, US5990349, WO9408957 and JP19660043158. Apart from the fact that these methods of preparation are not part of a process of sustainable development, these chemical methods have the disadvantage of producing a separate equal mixture of two enantiomers L and D.

[0005] totally organic syntheses by bacterial fermentation have been proposed in the literature with the advantage of producing only the L-enantiomer of methionine, as described for example in international applications

WO07077041, WO09043372, WO10020290 and WO10020681. Nevertheless, the absence of industrial production on a large scale to date, suggests that performance and / or production costs of these processes are still inadequate.

[0006] chemical / biological processes mixed just been successfully developed jointly by the company CJ Cheil-Jedang and the Applicant, in which a precursor of L-methionine is produced by bacterial fermentation and then enzymatically reacts with methyl mercaptan to producing exclusively L-methionine (see WO2008013432 and / or WO2013029690). These methods although high performance require the synthesis of methyl mercaptan site, which itself requires the synthesis of hydrogen by steam reforming of methane, the synthesis of hydrogen sulfide by hydrogenation of the sulfur and its synthesis from methanol and hydrogen sulphide, c '

[0007] There therefore remains a need for producing L-methionine by a mixing process wherein the equipment required for the synthesis of methyl mercaptan will be less than for a synthesis from hydrogen, hydrogen sulfide and methanol. It is in this context that the present invention takes place.

[0008] The present invention provides in effect replacing the methyl mercaptan in the process summarized below (WO2008013432 and / or WO2013029690) with dimethyl disulfide (DMDS):

MeSH

O-Acélyihomosénne fermentation Catalysis enzymattque

L-Methionine Step 1 (OAHS) £ ape 2

[0009] The methyl mercaptan (MeSH) is here used directly in the second step. The present invention proposes to substitute the methyl mercaptan by the enzymatic hydrogenolysis product of dimethyl disulfide in a preliminary step or combine together in a "one pot" reaction, in which glucose and DMDS produce L-methionine.

[0010] Regarding the synthesis of methyl mercaptan from dimethyl disulfide, can be found in the prior art the following items.

[0011] The EP0649837 patent application proposes a methyl mercaptan synthesis process by catalytic hydrogenolysis, with transition metal sulfides, the dimethyl disulfide with hydrogen. This method, although effective, requires relatively high temperatures of the order of 200 ° C to obtain industrially interesting productivities.

[0012] The skilled person also knows that it is possible to prepare methyl mercaptan by acidification of an aqueous solution of sodium methyl mercaptide (ChbSNa). This method has the major drawback of generating large amounts of salts such as sodium chloride or sodium sulphate, depending on whether hydrochloric acid is used or sulfuric acid. The aqueous salt solutions are often very difficult to treat and traces of malodorous products remaining are that this method is hardly feasible industrially.

[0013] It has now been found that could prepare the methyl mercaptan by enzymatic reduction of dimethyl disulfide (DMDS) during a preliminary step in the synthesis of L-methionine and it has also been found, surprisingly that we could achieve this enzymatic reduction of DMDS during the synthesis of L-methionine.

[0014] Thus, the present invention does relates to a process for preparing L-methionine similar to that proposed in international applications WO2008013432 and / or WO2013029690 and allows to overcome or at least to decrease, handling methylmercaptan, generating said methyl mercaptan in an enzymatically catalyzed reaction of DMDS, just before the use of said methyl mercaptan in the synthesis of methionine or generating said methyl mercaptan in an enzymatically catalyzed reaction of DMDS in situ in the reactor synthesis of L-methionine.

[0015] More particularly, the present invention has as its first object the method for preparing L-methionine, comprising at least the steps of:

a) preparing a mixture comprising:

1) dimethyl disulfide (DMDS)

2) a catalytic amount of amino acid carrying a thiol group or thiol group to peptide,

3) a catalytic amount of enzyme catalyzing the reaction of disulfide bond reducing said amino acid carrying a thiol group or thiol group of said peptide,

4) a reducing organic compound in a stoichiometric amount relative to the disulphide, particularly DMDS,

5) a catalytic amount of enzyme catalyzing the dehydrogenation reaction of the reducing organic compound of interest,

6) a catalytic amount of a cofactor common to the two enzymes of the catalyst system (dehydrogenase and reductase),

b) conduct the enzyme reaction to form methyl mercaptan (CH3-S), c) adding a precursor of L-methionine and converting said precursor with methyl mercaptan formed in step b) and

d) recovering and optionally purifying the formed L-methionine.

[0016] The components of step a) above can be added in different orders (the order of addition in step a) is not restrictive). In one embodiment of the invention, the amino acid carrying a thiol group and / or peptide bearing a thiol group may be in the form of disulfide said amino acid and / or said peptide, respectively, e.g. glutathione as glutathione disulfide.

[0017] In general, the enzyme catalyzing the reduction of the disulfide bridge formed between two equivalents of said amino acid carrying a thiol group or thiol group of said peptide is a reductase enzyme. The term "reductase" is used in the following description for the explanation of the present invention. Similarly, the enzyme catalyzing the dehydrogenation of organic reducing compound involved in step b) is generally referred to as dehydrogenase enzyme, the term "dehydrogenase" being selected from the following description for the explanation of the present invention.

[0018] Of the common cofactors for enzymes catalyzing both reduction and dehydrogenation (reductase and dehydrogenase) include as non-limiting examples cofactors flavin, nicotine and cofactors. We prefer to use the cofactor nicotinic especially the nicotinamide adenine dinucleotide (NAD), or better yet the nicotinamide adenine dinucleotide phosphate (NADPH). Cofactors listed above are advantageously used

under reduced forms thereof (e.g., NADPH, H +) and / or their oxidized forms (for example NADP +), that is to say, they may be added in these reduced forms and / or oxidized, in the reaction medium.

[0019] The organization and order of additions of components 1) to 6) in step a) can be achieved in different ways. The enzymatic reaction in step b) is initiated by the addition of one of the components of the catalytic system of the mixture of step a): either an enzyme or one of the compounds added in stoichiometric quantity (or disulfide reducing organic compound ) is one of the compounds added in a catalytic amount (amino acid carrying a thiol group or thiol group to the peptide or disulfide corresponding to said thiol or said peptide or alternatively cofactor).

[0020] Thus and according to one embodiment of the present invention the method for preparing L-methionine, comprises at least the steps of:

a ') preparing a mixture comprising:

• dimethyl disulfide (DMDS)

• a catalytic amount of amino acid carrying a thiol group or thiol group to peptide,

• a catalytic amount of reductase enzyme corresponding to said amino acid carrying a thiol group or thiol group in said peptide,

• a catalytic amount of NADPH

b ') adding an organic reducing compound in stoichiometric amount relative to the dimethyl disulphide) with a catalytic amount of the corresponding dehydrogenase enzyme,

c ') carrying out the enzyme reaction to form methyl mercaptan (CH 3 SH), d') converting a precursor of L-methionine with the methyl mercaptan formed in step c '), and

e ') recovering and optionally purifying the formed L-methionine.

[0021] According to the method of the invention, methyl mercaptan, generally formed in a gaseous state, is then directly contacted with a methionine precursor as described hereinafter.

[0022] The method of synthesis of L-methionine according to the invention is first based on the enzymatic reduction of dimethyl disulfide with a reducing organic compound, which is a hydrogen donor as will be defined later according to the following reaction using glucose as the reducing organic compound (hydrogen donor):

DMDS Glucose MeSH Gluconolacton

[0023] It has now been discovered that this reaction is readily catalyzed by the enzyme system implementing a thiol group to amino acid or a thiol group in the peptide, e.g., glutathione, as a complex (amino acid or peptide) / corresponding reductase enzyme, regenerated by the organic hydrogen donor compound, as described in Figure 1 herein.

[0024] Thus according to the Figure 1 illustration, the peptide ( "glutathione" shown) reduces the disulfide ( "DMDS" shown) mercaptan ( "methyl" shown), by transforming into disulfide-bonded peptide ( "disulfide glutathione "shown). The reductase enzyme ( "glutathione reductase" shown, EC 1 .8.1 .7 or EC 1 .6.4.2) regenerates the peptide (glutathione), while oxidizing the cofactor ( "NADPH, H +" shown). The oxidized form ( "NADP +" shown) is then reduced using an oxidation-reduction enzyme complex, called "recycle", well known to those skilled in the art and comprising the dehydrogenase enzyme involved ( "glucose dehydrogenase "shown with the example of enzyme classification number EC 1 .1 .1 .47), and the reducing organic molecule (" glucose "shown).

[0025] More particularly, the peptide (example shown glutathione) reduced dimethyl disulfide to methyl mercaptan by transforming into disulfide-bonded peptide (shown glutathione disulfide). The reductase enzyme ( "glutathione reductase" shown, EC 1 .8.1 .7 or EC 1 .6.4.2) regenerates the peptide (glutathione) and the same enzyme is regenerated by a redox enzyme complex known to those skilled the art, such as NADPH / NADP + complex (nicotine adenine dinucleotide phosphate (reduced form and oxidized form)). In turn NADP + to NADPH is regenerated through the corresponding dehydrogenase enzyme to the organic compound employed reducer (here the "glucose

dehydrogenase "EC 1 .1 .1 .47) by said organic reducing compound (shown glucose) that supplies hydrogen (hydrogen donor) by turning into its oxidized form (here gluconolactone).

[0026] According to one embodiment particularly adapted, the system glutathione / glutathione disulfide associated with the glutathione reductase enzyme of the present invention allows to reduce the DMDS methyl mercaptan.

[0027] Glutathione is a tripeptide widely used in biology. This species in reduced form (GSH) and oxidized (glutathione disulfide) forms an important redox couple in the cells. So glutathione is vital to remove heavy metals organizations. For example, the application WO05107723 describes a formulation in which glutathione is used to form a chelate preparation, patent US4657856 teaches that glutathione also used to destroy peroxides such ΙΉ2Ο2 H2O via glutathione peroxidase. Finally glutathione also reduces disulfide bonds in proteins (Rona Chandrawati, "Triggered Cargo Release Encapsulated by Enzymatic Catalysis in Capsosomes" Nano Lett (1 201), Vol. 1 1, 4958-4963).

[0028] According to the method of the invention, a catalytic amount of amino acid carrying a thiol group or thiol group to peptide, is implemented for the production of methyl mercaptan from dimethyl disulphide.

[0029] Among the amino acids thiol group of carriers used in the process of the present invention include, by way of non-limiting examples the cysteine ​​and homocysteine. The redox enzyme systems used can regenerate the catalytic cycle in the same way, these cases are in the system cysteine ​​/ cystine reductase EC 1 .8.1 .6, homocysteine ​​and / homocysteine ​​reductase.

[0030] It may be advantageous to use homocysteine ​​as this amino acid may be prepared from OAHS (precursor of L-methionine), hydrogen sulfide (H2S) and the enzyme methionine , that is to say, the enzyme catalyzing the reaction leading to methionine. Thus, a very small amount of S h in the reaction medium creates, in situ, the cycle equivalent to that of glutathione.

[0031] Among the thiol group of bearing peptides used in the process of the present invention include, by way of non-limiting examples glutathione and thioredoxin. Glutathione system / glutathione reductase described above, may

thus be replaced by the thioredoxin system (CAS no. 52500-60-4 ythiorédoxine reductase (EC 1 .8.1 .9 or EC 1 .6.4.5).

[0032] The glutathione and glutathione system / glutathione reductase are especially preferred for the present invention, due to the ease of supply and the costs thereof.

[0033] Among the reducing organic compounds which can be used in the context of the present invention, the hydrogen donor compounds are particularly preferred, and among these, the compounds are well suited organic reducing compounds donors 'holders hydrogen hydroxyl function, such as alcohols, polyols, sugars, and others.

[0034] The enzyme used is an enzyme capable of dehydrogenating the hydrogen compound, for example an alcohol dehydrogenase. Glucose is a sugar particularly well suited to be implemented in the method of the present invention with glucose dehydrogenase to yield the gluconolactone.

[0035] In the method according to the invention, in the case where the enzymatic reduction of DMDS is carried out in a separate reactor for the synthesis of L-methionine, only the glucose is used in stoichiometric amount, all other components (glutathione cofactor (e.g., NADPH) and the two enzymes) are used in catalytic amount. In case the reaction of DMDS the enzymatic reduction is done with the synthesis of L-methionine in a single reactor said "one pot", the precursor of L-methionine is added in stoichiometric amount, while the additional reactants this synthesis such as pyridoxal phosphate (PLP) and specific to this enzyme reaction are added in catalytic amounts.

[0036] The concentrations of pyridoxal phosphate and specific enzyme preferred precursor are those we can find in international applications WO2008013432 and / or WO2013029690.

[0037] The benefits synthesizing by enzymatic catalysis of methyl mercaptan from dimethyl disulphide are numerous, both in the case of two successive steps of the method or "one pot". These benefits include the ability to work in aqueous or aqueous-organic solution, under very mild conditions of temperature and pressure and pH conditions close to neutrality. All these conditions are typical of a process called "green" or "sustainable" and are completely compatible with the preparation of L-methionine, as described in international applications WO2008013432 and / or WO2013029690.

[0038] Another advantage when the process uses dimethyl disulfide is as methyl product, which is in the gaseous state under the reaction conditions, leaves the reaction medium as and when it is formed. Methyl mercaptan can be used directly at the outlet of the reactor in the synthesis of L-methionine, as described for example in WO2008013432 and / or WO2013029690, that is to say from e.g., O-acetylhomoserine or O-succinylhomoserine and enzymes such as O-acetylhomoserine sulfhydrylase or Ο-succinylhomoserine sulfhydrylase respectively.

[0039] Methyl mercaptan can also be easily liquefied cryogenic example if you want to isolate. can possibly accelerate leaving the reaction medium by bubbling by introducing a slight flow of inert gas, preferably nitrogen.

[0040] The exit gas containing nitrogen and methyl mercaptan may, if desired and if necessary, be recycled into the first reactor (enzymatic reduction of DMDS) after passing through the second reactor (synthesis of L-methionine) if methyl mercaptan was not completely converted to L-methionine. The method of the invention thus discloses a method for synthesizing L-methionine in two successive enzymatic steps from a precursor of L-methionine and DMDS.

[0041] It is also possible to carry out the synthesis of L-methionine in a single reactor. In this case is added to the enzyme reduction system of DMDS (step a) above) all reagents required for the synthesis of L-methionine and the reactor is closed to prevent the departure of the methyl mercaptan formed by enzymatic reduction in situ DMDS. Methyl mercaptan reacts with the precursor L-methionine to give L-methionine. The method according to the present invention thus discloses a direct synthesis process of L-methionine from a precursor of L-methionine and DMDS, as shown in Figure 2 attached, wherein the synthesis from OAHS, DMDS and glucose.

[0042] Dimethyl disulphide (DMDS) can be produced on a different site from methyl mercaptan and an oxidant such as oxygen, sulfur or oxygenated water for example, or from dimethyl sulfate and sodium disulfide. DMDS can also come from a source of "disulfide Oils" (DSO) purified for example by reactive distillation as described in WO2014033399 application.

[0043] The reduction in enzymatic catalysis of DMDS can be considered as a method to prevent the transportation of methyl mercaptan production site by industrial existing channels, to its site of use, if they are different. In fact, methyl mercaptan is a gas at room temperature, highly toxic and smelly which greatly complicates its transportation already highly regulated unlike DMDS. Thus DMDS can be used to produce methyl mercaptan directly on the site of use thereof in the synthesis of L-methionine, thereby further reducing the disadvantages of toxicity and odor of the product, and industrial risks attached thereto.

[0044] In the case of the synthesis process in two successive steps, DMDS being consumed in the reaction and the outgoing methyl mercaptan from the reaction medium as and when it is formed, only the dehydrogenation of organic compound product reducer, e.g. gluconolactone, accumulates in the reaction medium, in the case of a continuous feed of glucose and DMDS. When gluconolactone concentration exceeds the saturation in the reaction conditions, the latter will precipitate and can then be isolated from the reaction medium by any means known in the art.

[0045] The gluconolactone can have multiple uses. It is for example used as a food additive known by the acronym E575. Gluconolactone hydrolyzes in aqueous acids to form gluconic acid also used as food additive (E574). Gluconolactone is also used for tofu production (see CN103053703) for the food industry.

[0046] The gluconolactone can in particular and advantageously, in the sense that it represents the "waste" of the method according to the present invention, replace glucose in any fermentation reaction to produce either bioethanol or other sugar fermentation of a molecule or starch.

[0047] Some bacteria may be using fermentation gluconolactone as a carbon source, as described by JP van Dijken, "Novel pathway for alcoholic fermentation of yeast Saccharomyces gluconolactone in the bulderi" J. Bacteriol. (2002), Vol . 184 (3), 672-678.

[0048] An obvious advantage of the gluconolactone in the process according to the invention is recycled to the synthesis of the precursor of L-methionine. Indeed this synthesis being a bacterial fermentation using glucose, gluconolactone could easily replace some of this glucose. This recycling can in these conditions represent a significant economic advantage.

[0049] Even in the case where the reaction is carried out under the conditions "one pot" defined above, and gluconolactone being much more soluble than L-methionine, it is easy to separate from the reaction medium according to conventional techniques and well known to those skilled in the art.

[0050] Other sugars may still be used in the process of the invention, and for example it is possible to replace glucose system / gluconolactone / glucose dehydrogenase by the following system: glucose-6-phosphate / 6-phosphoglucono -5-lactone dehydrogenase / Glucose-6-phosphate (EC 1 .1 .1 .49).

[0051] It is also possible in the method of the invention to use a sugar substitute in alcohol, and thus be used in place of glucose system / glucono-lactone / glucose dehydrogenase, the following general system: alcohol / ketone or aldehyde / alcohol dehydrogenase (EC 1 .1 .1) and more particularly the system isopropanol / acetone / isopropanol dehydrogenase (EC 1 .1 .1 .80).

[0052] In fact, this system allows to obtain, from DMDS and isopropanol, a mixture of methyl mercaptan (MeSH) and acetone exiting the reaction medium (so no accumulation of any product) . The MeSH and acetone can be easily separated by simple distillation if desired.

[0053] According to one embodiment, the method according to the invention comprises the preparation by enzymatic reduction of DMDS and then reacting the methyl mercaptan formed with a precursor of L-methionine to give L-methionine. In this case, the method according to the invention comprises at least the following steps:

Step 1: preparing a precursor of L-methionine, for example, by bacterial fermentation of glucose (see WO2008013432 and / or WO2013029690) Step 2: Enzymatic reduction of DMDS in a reactor R1 with methyl mercaptan out of forming said reactor R1 (corresponding to steps a ') to c') above),

Step 3: Enzymatic synthesis of L-methionine in a reactor R2 with the precursor of step 1 and methyl mercaptan in Step 2 (corresponding to step d ') above),

Step 4 (optional): recycling gluconolactone formed in Step 3 in Step 1,

Step 5: Recovery and optionally purification of the formed L-methionine (corresponding to step e ') above).

[0054] In Step 1, we find the field conditions used in the following patents (see WO2008013432 and / or WO2013029690).

[0055] For Step 2, the reaction temperature is within a range from 10 ° C to 50 ° C, preferably between 15 ° C and 45 ° C, more preferably between 20 ° C and 40 ° C.

[0056] The pH of the reaction may be between 6 and 8, preferably between 6.5 and 7.5. The pH of the reaction medium may be adjusted using a buffer. So most preferably be selected, for example, pH 0.1 phosphate buffer rnol.L "1 of to 7.3.

[0057] The pressure used for the reaction can range from a reduced pressure relative to the atmospheric pressure to several bars (several hundred kPa), depending on the reagents used and the equipment used. Reduced pressure may indeed allow a quicker degassing of methyl mercaptan formed but has the disadvantage of increasing saturation vapor pressure of water and DMDS, polluting a little methyl mercaptan formed. Preferably, a pressure from atmospheric pressure will be used at 20 bar (2 MPa) and more preferably it will work under a pressure ranging from atmospheric pressure to 3 bars (300 kPa).

[0058] In Step 3, reference is made to the international application WO2013029690 for ideal conditions with the potential difference of introducing a flow of nitrogen in the reactor R1 to arrive in the reactor R2 and recycle these gases from the reactor R2 to the reactor R1 at the desired pressure if methylmercaptan has not completely reacted in the reactor R2.

[0059] According to another embodiment (another variant), the method according to the present invention is carried out in a single reactor ( "one pot"), and in this case comprises at least the following steps:

Step 1 ': preparing a precursor of L-methionine by bacterial fermentation of glucose for example (similar to Step 1 above),

Step 2 ': enzymatic reduction of DMDS in a reactor R1 with in situ formation of methyl mercaptan and joint enzymatic synthesis of L-methionine in the same reactor with the precursor obtained in step V,

Step 3 (optional): recycling gluconolactone formed in step 2 'in the step 1', and

Step 4 ': recovery and optionally purification of the formed L-methionine.

[0060] In Step V, we find the field conditions used in international applications WO2008013432 and / or WO2013029690.

[0061] In Step 2 ', the operating conditions are as follows.

[0062] The reaction temperature is within a range from 10 ° C to

50 ° C, preferably 15 ° C to 45 ° C, preferably from 20 ° C to 40 ° C.

[0063] The pH of the reaction is advantageously between 6 and 8, preferably between 6.2 and 7.5. So Most preferably, the reaction is carried out at the pH of phosphate buffer 0.2 rnol.L "1 and equal to 7.0.

[0064] Preferably, the method is performed at a pressure ranging from atmospheric pressure to 20 bar (2 MPa) and more preferably from atmospheric pressure to 3 bars (300 kPa).

[0065] The molar ratio DMDS / L-methionine precursor is between 0.1 and 10, usually between 0.5 and 5, and preferably the molar ratio is the stoichiometry (molar ratio = 0.5) but can be superior if this is beneficial for the kinetics of the reaction.

[0066] In one or other of the process according to the invention variants, it may be carried out batchwise or continuously in a reactor made of glass or metal depending on the operating conditions and reagents used.

[0067] In one or the other variants of the method according to the invention, the molar ratio of organic reducing compound / DMDS is ideal stoichiometry (molar ratio = 1) but may range from 0.01 to 100 if the skilled in the art there is any interest such as continuous addition of DMDS as the reducing compound is introduced from the beginning into the reactor. Preferably this molar ratio is selected between 0.5 and 5 globally on the entire reaction.

[0068] The elements present in a catalytic amount in the mixture prepared in step a) above (amino acid carrying a thiol group or thiol group to peptide or disulfide corresponding to said amino acid or disulfide corresponding to said peptide, reductase enzyme, dehydrogenase enzyme, cofactor, eg NADPH) are readily commercially available or may be prepared according to techniques well known to those skilled in the art. These different elements may be in solid or liquid form and may most preferably be dissolved in water to be used in the method of the invention. The enzymes used can also be grafted on a support (if the supported enzymes).

[0069] The aqueous solution of enzyme complex comprising the amino acid or peptide can also be reconstituted by methods known to those skilled in the art, for example by permeabilization of cells which contain these elements. This aqueous solution having a composition given in Example 1 the following may be used in weight contents of between 0.01% and 20% based on the total weight of the reaction medium. Preferably we use a content of between 0.5% and 10%.

[0070] the invention will be better understood with the following non-limiting examples in relation to the scope of the invention.

EXAMPLE 1: A process in two successive steps

[0071] In a reactor R1 containing 150 mL of phosphate buffer 0.1 mol / L at pH 7.30, were introduced 10 ml of enzyme glutathione complex (Aldrich) and 19.2 g (0.1 mol) glucose. The enzyme complex solution contains: 185 mg (0.6 mmol) of glutathione, 200 U of glutathione reductase, 50 mg (0.06 mmol) of NADPH and 200 U of glucose dehydrogenase. The reaction medium is brought to 25 ° C with mechanical stirring. A first transaction is made t = 0. Thereafter, the dimethyl disulfide (9.4 g, 0.1 mol) was placed in a buret and added dropwise to the reactor, the reaction begins. A nitrogen stream is placed in the reactor.

[0072] An analysis by gas chromatography of gas leaving the reactor shows almost essentially the presence of nitrogen and methyl (traces of water). These output gases are sent into the reactor R2. The

DMDS is introduced at 6 hours in the reactor R1. A final analysis by gas chromatography of the reaction medium of the reactor R1 confirms the absence of DMDS, and analyzed by UPLC / mass shows traces of glucose and the almost exclusive presence of gluconolactone (gluconic acid traces).

[0073] In parallel, in the second reactor R2 containing 75 mL of phosphate buffer 0.1 mol L "1 at pH 6.60, were introduced 5 g of O-acetyl-L-homoserine (OAHS) (Ο- acetylhomoserine was synthesized from L-homoserine and acetic anhydride according Sadamu Nagai, "Synthesis of O-acetyl-homoserine-i", Academic Press, (1971), vol.17, p. 423-424). The solution is heated to 35 ° C with mechanical stirring.

[0074] Before commencement of the reaction, a sample (t = 0) 1 mL of the reaction medium is performed. A pyridoxal phosphate solution (1, 6 mmol, 0.4 g) and the enzyme O-acetyl-L-homoserine sulfhydrylase (0.6 g) are dissolved in 10 mL of water and then added to the reactor.

[0075] Methyl mercaptan is introduced via the reaction of the reactor R1 and driven by a stream of nitrogen. The reaction begins. The formation of L-methionine and the disappearance of the OAHS followed by HPLC. The gas outlet of the reactor R2 are trapped in an aqueous sodium hydroxide solution (sodium hydroxide) at 20%. Analyzes show that the OAHS was converted to 52% in L-methionine and that excess DMDS was converted to methyl mercaptan found in the trap soda.

EXAMPLE 2 Process "one pot"

[0076] In a reactor containing 150 ml of phosphate buffer 0.2 mol L "1 at pH 7, are introduced 10 mL of the enzyme complex, 6 g (33 mmol) of glucose and 5 g (31 mmol) of O -acetyl-L-homoserine (OAHS, ΓΟ-acetyl-L-homoserine was synthesized from L-homoserine and acetic anhydride according Sadamu Nagai, "Synthesis of O-acetyl-L-homoserine," Academic Press, ( .. 1971), vol.17, pp 423-424) the enzyme of the complex solution contains: 185 mg (0.6 mmol) of glutathione, 200 U of glutathione reductase, 50 mg (0.06 mmol) of NADPH, 200 U glucose dehydrogenase, 0.4 g (1, 6 mmol) of pyridoxal phosphate and 0.6 g of O-acetyl-L-homoserine sulfhydrylase.

[0077] The reaction medium is brought to 27 ° C with mechanical stirring. A first sample t = 0 is made. Thereafter dimethyl disulfide (3 g, 32 mmol) was placed in a buret and added dropwise to the reactor which was sealed to prevent any release of methyl mercaptan, the reaction begins. The reaction was followed by HPLC to see the disappearance of the OAHS and formation of L-methionine. After 6 hours, 21% of OAHS have been converted into L-methionine showing the ability to produce L-methionine by a method "one-pot" from a precursor of L-methionine, DMDS and a reducing organic compound.

CLAIMS

1. A process for preparing L-methionine, comprising at least the steps of: a) preparing a mixture comprising:

1) dimethyl disulfide (DMDS)

2) a catalytic amount of amino acid carrying a thiol group or thiol group to peptide,

3) a catalytic amount of enzyme catalyzing the reaction of disulfide bond reducing said amino acid carrying a thiol group or thiol group of said peptide,

4) a reducing organic compound in a stoichiometric amount relative to the disulphide, particularly DMDS,

5) a catalytic amount of enzyme catalyzing the dehydrogenation reaction of a reducing organic compound concerned,

6) a catalytic amount of a cofactor common to the two enzymes of the catalyst system (dehydrogenase and reductase),

b) conduct the enzyme reaction to form methyl mercaptan (CH3-S), c) adding a precursor of L-methionine and converting said precursor with methyl mercaptan formed in step b) and

d) recovering and optionally purifying the formed L-methionine.

2. The method of claim 1, comprising the steps of at least:

a ') preparing a mixture comprising:

• dimethyl disulfide (DMDS)

• a catalytic amount of amino acid carrying a thiol group or thiol group to peptide,

• a catalytic amount of reductase enzyme corresponding to said amino acid carrying a thiol group or thiol group in said peptide,

• a catalytic amount of NADPH

b ') adding an organic reducing compound in stoichiometric amount relative to the dimethyl disulphide) with a catalytic amount of the corresponding dehydrogenase enzyme,

c ') carrying out the enzyme reaction to form methyl mercaptan (CH 3 SH), d') converting a precursor of L-methionine with the methyl mercaptan formed in step c '), and

e ') recovering and optionally purifying the formed L-methionine.

3. The method of claim 1 or claim 2, wherein methyl mercaptan is directly contacted with a methionine precursor.

4. A method according to any preceding claim, wherein the reducing organic compound is an organic compound gear carrier hydrogen donor hydroxyl function, chosen from alcohols, polyols, sugars.

5. A method according to any preceding claim, wherein the reducing organic compound is selected from glucose, glucose 6-phosphate and isopropanol.

6. A method according to any preceding claim, wherein the thiol-bearing amino acid or thiol group of carrier peptide is selected from cysteine, homocysteine, glutathione and thioredoxin.

7. A method according to any preceding claim, wherein the precursor of L-methionine is selected from O-acetyl-L-homoserine and O-succinyl-L-homoserine.

8. A method according to any preceding claim, wherein methyl mercaptan is used directly at the outlet of the reactor in the synthesis of L-methionine.

9. The method of claim 8, comprising at least the following steps Step 1 Preparation of a precursor of L-methionine, for example, by bacterial fermentation of glucose,

Step 2: Enzymatic reduction of DMDS in a reactor R1 with methyl mercaptan out of forming said reactor R1,

Step 3: Enzymatic synthesis of L-methionine in a reactor R2 with the precursor of step 1 and methyl mercaptan in Step 2,

Step 4 (optional): recycling gluconolactone formed in Step 3 in Step 1,

Step 5: Recovery and optionally purification of the formed L-methionine.

10. A method according to any one of claims 1 to 7, wherein the synthesis of methyl mercaptan from DMDS and synthesis of L-methionine from said methyl mercaptan is conducted in a single reactor.

11. The method of claim 10, comprising at least the steps of:

Step 1 ': preparing a precursor of L-methionine by bacterial fermentation of glucose,

Step 2 ': enzymatic reduction of DMDS in a reactor R1 with in situ formation of methyl mercaptan and joint enzymatic synthesis of L-methionine in the same reactor with the precursor obtained in step 1',

Step 3 (optional): recycling gluconolactone formed in step 2 'in the step 1', and

Step 4 ': recovery and optionally purification of the formed L-methionine.

12. A method according to any preceding claim, carried out batchwise or continuously.

13. A method according to any preceding claim, wherein the molar ratio of organic reducing compound / DMDS ideal ranges from 0.01 to 100, preferably said molar ratio is selected between 0.5 and 5, and most Most preferably, the molar ratio is 1.

14. A method according to any preceding claim, wherein the molar ratio DMDS / L-methionine precursor is between 0.1 and 10, usually between 0.5 and 5, and preferably the molar ratio is the stoichiometry (molar ratio = 0.5).

15. A method according to any preceding claim, wherein the reaction temperature is within a range from 10 ° C to 50 ° C, preferably 15 ° C to 45 ° C, preferably from 20 ° C to 40 ° C.