METHOD FOR PRODUCING L-METHIONINE

Abstract

Provided is an enzymatic process for preparing L-methionine from diethyl disulfide.

Claims

1-14. (canceled)

15. A process for the preparation of L-methionine, comprising: (a) preparing a mixture, comprising: (1) dimethyl disulfide (DMDS), (2) a catalytic amount of an amino acid bearing a thiol group or of a thiol-group-containing peptide, wherein the amino acid bearing a thiol group or the thiol-group-containing peptide may optionally be in the form of the corresponding disulfide, (3) a catalytic amount of a reductase enzyme catalyzing the reaction for the reduction of a disulfide bridge of the amino acid bearing a thiol group or of the thiol-group-containing peptide, (4) hydrogen, (5) a catalytic amount of dehydrogenase enzyme catalyzing the reaction for the reduction of hydrogen, and (6) a catalytic amount cofactor common to the reductase and the dehydrogenase, (b) carrying out the enzymatic reaction to form methyl mercaptan (CH.sub.3SH), (c) adding a precursor of L-methionine and reacting the precursor with the methyl mercaptan formed in (b) to produce L-methionine, and (d) recovering and, optionally, purifying the L-methionine.

16. The process of claim 15, comprising: (a) preparing a mixture, comprising: dimethyl disulfide (DMDS), a catalytic amount of amino acid bearing a thiol group or of a thiol-group-containing peptide, wherein the amino acid bearing a thiol group or the thiol-group-containing peptide may optionally be in the form of the corresponding disulfide, a catalytic amount of a reductase enzyme corresponding to the amino acid bearing a thiol group or to the thiol-group-containing peptide, and a catalytic amount of NADPH, (b) adding hydrogen with a catalytic amount of a hydrogen dehydrogenase enzyme, (c) carrying out the enzymatic reaction to form methyl mercaptan (CH.sub.3SH), (d) converting an L-methionine precursor with the methyl mercaptan formed in (c) to produce L-methionine, and (e) recovering and, optionally, purifying the L-methionine.

17. The process of claim 15, wherein the methyl mercaptan is directly placed in contact with the precursor of L-methionine.

18. The process of claim 15, wherein the amino acid bearing a thiol group or the peptide bearing a thiol group is chosen from cysteine, homocysteine, glutathione and thioredoxin.

19. The process of claim 15, wherein the L-methionine precursor is chosen from O-acetyl-L-homoserine and O-succinyl-L-homoserine.

20. The process claim 15, in which hydrogen is added to the reaction reaction medium via bubbling.

21. The process of claim 15, comprising preparing methyl mercaptan by enzymatically reducing the DMDS, then reacting the methyl mercaptan with an L-methionine precursor to produce L-methionine.

22. The process according of claim 21, comprising: (1) preparing an L-methionine precursor, (2) enzymatically reducing DMDS in a first reactor with formation of methyl mercaptan, and optionally, unconverted hydrogen, leaving the first reactor, (3) enzymatically synthesizing L-methionine in a second reactor with the L-methionine precursor from (1) and the methyl mercaptan from (2), (4) optionally, recycling of the unconverted hydrogen to (2) and recycling the methyl mercaptan to (2) or (3), and (5) recovering and, optionally, purifying the L-methionine.

23. The process of claim 15, wherein the synthesis of methyl mercaptan from DMDS and the synthesis of L-methionine from the methyl mercaptan are carried out in one and the same reactor.

24. The process of claim 23, comprising: (1) preparing an L-methionine precursor, (2) enzymatically reducing DMDS in a first reactor with in situ formation of methyl mercaptan and concomitant enzymatic synthesis of L-methionine in the same reactor with the precursor obtained in (1), (3) optionally, recycling loop of the hydrogen and methyl mercaptan in the first reactor, at (2), and (4) recovering and, optionally, purifying the L-methionine.

25. The process of claim 15, which is carried out batchwise or continuously.

26. The process of claim 15, wherein the hydrogen/DMDS molar ratio varies from 0.01 to 100 over the whole of the reaction.

27. The process of claim 15, wherein the hydrogen/DMDS molar ratio varies from 1 to 20 over the whole of the reaction.

28. The process of claim 15, wherein the DMDS/L-methionine precursor molar ratio is between 0.1 and 10.

29. The process of claim 15, wherein the DMDS/L-methionine precursor molar ratio is between 0.5 and 5.

30. The process of claim 15, wherein the reaction temperature is within a range extending from 10 C. to 50 C.

31. The process of claim 15, wherein the reaction temperature is within a range extending from 15 C. to 45 C.

32. The process of claim 15, wherein the reaction temperature is within a range extending from 20 C. to 40 C.

33. The process of claim 15, wherein the amino acid bearing a thiol group or a thiol-group-containing peptide is glutathione.

34. The process of claim 15, wherein the cofactor is NADPH.

Description

EXAMPLE 1: PROCESS IN 2 SUCCESSIVE STEPS

[0084] 10 ml of glutathione enzymatic complex (Aldrich) are introduced into a reactor R1 containing 150 ml of aqueous solution buffered to pH 7.8. The solution of enzymatic complex contains: 185 mg (0.6 mmol) of glutathione, 200 U of glutathione reductase, 50 mg (0.06 mmol) of NADPH and 200 U of hydrogen dehydrogenase enzyme. The reaction medium is brought to 35 C. with mechanical stirring. A first sample is taken at t=0, Subsequently, the dimethyl disulfide (9.4 g, 0.1 mol) is placed in a burette and added dropwise to the reactor.

[0085] At the same time, a 4 L.h.sup.1 stream, of hydrogen (measured under normal temperature and pressure conditions) is introduced into the reactor via bubbling. The reaction is carried out at atmospheric pressure.

[0086] Gas chromatography analysis of the gases leaving the reactor shows virtually essentially the presence of hydrogen and methyl mercaptan (some traces of water), The DMDS and the hydrogen (hydrogen/DMDS molar ratio over the, whole of the reaction=10.7) are introduced in 6 hours and a final gas chromatography analysis of the reaction medium confirms the absence of methyl mercaptan which has been driven out of the reactor by the excess hydrogen. These outlet gases from the reactor R1 are sent directly into the reactor R2.

[0087] In parallel, 5 g of O-acetyl-L-homoserine (OAHS) (the O-acetyl-L-homoserine was synthesized from L-homoserine and acetic anhydride as per Sadamu Nagai, Synthesis of O-acetyl-L-homoserine, Academic Press, (1971), vol. 17, pp. 423-424) are introduced into the second reactor R2 containing 75 ml of 0.1 mol.l.sup.1 phosphate buffer at pH 6.60. The solution is brought to 35 C. with mechanical stirring.

[0088] Before the reaction starts, a sample (t=0) of 1 ml of the reaction medium is taken. A solution of pyridoxal phosphate (10 mmol. 0.4 g) and the enzyme O-acetyl-L-homoserine sulfhydrylase (0.6 g) are dissolved in 10 ml of water then added to the reactor

[0089] The methyl mercaptan is introduced via the reaction of the reactor R1 and advantageously propelled by the excess hydrogen, or else when the hydrogen is in stoichiometric or substoichiometric, conditions with respect to the DMDS the methyl mercaptan is advantageously propelled by a stream of inert gas, for example a nitrogen stream. The reaction then begins. The formation of L-methionine and the disappearance of OAHS are monitored by HPLC. The outlet gases from the reactor R2 are trapped in a 20% aqueous sodium hydroxide solution. The analyses show that the OAHS has been converted to a degree of 52% into L-methionine and that the excess DMDS has been converted into methyl mercaptan found in the sodium hydroxide trap.

EXAMPLE 2: ONE POT PROCESS

[0090] 10 ml of the enzymatic complex, 5 g (31 mmol) of O-acetyl-L-homoserine (OAHS the O-acetyl-L-homoserine was synthesized from L-homoserine and acetic anhydride as per Sadamu Nagai, Synthesis of O-acetyl-l-homoserine, Academic Press, (1971), vol. 17, pp. 423-424) are introduced into a reactor containing 150 ml of 0.2 mol.l.sup.1 phosphate buffer at pH 7. The solution of the enzymatic complex contains: 185 mg (0.6 mmol) of glutathione, 200 U of glutathione reductase, 50 mg (0.60 mmol) of NADPH, 200 U of hydrogen dehydrogenase enzyme, 0.4 g (1.6 mmol) of pyridoxal phosphate and 0.6 g of O-acetyl-L-homoserine sulfhydrylase.

[0091] The reaction medium is brought to 35 C. with mechanical stirring. A first sample at t=0 is taken. Subsequently, the dimethyl disulfide (3 g, 32 mmol) is placed in a burette and added dropwise and a flow of 4 litres/h of hydrogen is introduced; the reaction begins The reaction is monitored by HPLC to see the disappearance of the OAHS and the formation of the L-methionine. After 6 hours, 12% of the OAHS has been converted into L-methionine, demonstrating the possibility of producing L-methionine by a one pot process from an L-methionine precursor, DMDS and hydrogen.

EXAMPLE 3: ONE POT PROCESS

[0092] 10 ml of the enzymatic complex, 5 g (31 mmol) of O-acetyl-L-homoserine (OAHS the O-acetyl-L-homoserine was synthesized from L-homoserine and acetic anhydride as per Sadamu Nagai, Synthesis of O-acetyl-L-homoserine, Academic Press, (1971), vol. 17, pp. 423-424) are introduced into a reactor containing 70 ml of 0.1 mol.l.sup.1 phosphate buffer at pH 6.8.

[0093] The solution of the enzymatic complex contains: 200 mg (0.65 mol) of glutathione, 500 U of glutathione reductase, 100 mg (0.13 mol) of NADPH, 50 U of hydrogen dehydrogenase, 400 mg (1.6 mmol) of pyridoxal phosphate, 2 g of O-acetyl-L-homoserine and 0.6 g of O-acetyl-L-homoserine sulfhydrylase.

[0094] The hydrogen dehydrogenase is obtained from the culture of microorganisms (according to Biller et al., Fermentation Hyperthermophiler Mikroorganismen am Beispiel von Pyrococcus Furiosus, Shaker Verlag, Maastricht/Herzogenrath, 2002), using techniques well known to those skilled in the art.

[0095] The reaction medium is brought to 35 C. with mechanical stirring and nitrogen flushing. A first sample is taken at t=0. Subsequently, 20 g (0.22 mol) of dimethyl disulfide are added by means of a syringe. At the same time, 4 l.h.sup.1 of hydrogen (measured under standard conditions of temperature and pressure) are introduced by bubbling into the reaction medium. The reaction started in this way is carried out at atmospheric pressure for 18 hours. The reaction is monitored by HPLC to see the disappearance of the OAHS and the formation of L-methionine. At the end of the reaction, 27% of the OAHS has been converted into L-methionine, demonstrating the possibility of producing L-methionine by a one pot process from an L-methionine precursor, DMDS and hydrogen.