METHIONINE PRODUCTION

Abstract

There is provided a method of producing a method of producing methionine, the method comprising contacting vinylglycine or derivatives thereof with at least one free radical methyl mercaptan in a reaction medium.

Claims

1. A method of producing methionine, the method comprising contacting vinyiglycine or derivatives thereof with at least one free radical methyl mercaptan in a reaction medium.

2. The method according to claim 1, wherein the ratio of methyl mercaptan to vinylglycine or derivatives thereof is 1:1-1:10.

3. The method according to claim 1, wherein the free radical methyl mercaptan is formed by contacting methyl mercaptan with at least one free radical initiator in the reaction medium.

4. The method according to claim 3, wherein the free radical initiator is selected from the group consisting of azobisisobutyronitrile (AIBN), N-bromosuccinimide (NBS), dibenzoyl peroxide (DBPO) and 2,2-Azobis (2-(2-imidazolin-2-yl)propane) dihydrochloride.

5. The method according to claim 3, wherein the ratio of free radical initiator to methyl mercaptan is selected from the range of 1:10000 to 1:10.

6. The method according to claim 3, wherein the free radical initiator is dibenzoyl peroxide (DBPO).

7. The method according to claim 1, wherein the free radical methyl mercaptan is formed by contacting methyl mercaptan with UV light.

8. The method according to claim 7, wherein the UV light has a wavelength of 300 nm.

9. The method according to claim 1, wherein the free radical methyl mercaptan is formed by contacting methyl mercaptan with at least one photoinitiator and UV light at a wavelength of 365 nm.

10. The method according to claim 9, wherein the photoinitiator is selected from the group consisting of hydroxylcyclohexyl phenyl ketone (HCPK), 2-benzyl-2-N, N-dimethylamino-1-(4-morpholino phenyl)-1-butanone (DBMP), 1-hydroxyl cyclohexyl phenyl ketone, and beozophenone, 2-methyl-1-(4-methylthio)phenyl-2-morpholino propan-1-onc (MMP).

11. The method according to claim 1, wherein the vinylglycine or derivatives thereof is formed from: (a) contacting glutamic acid with a genetically modified cell, wherein the cell comprises at least a first genetic mutation that increases the expression relative to the wild type cell of an enzyme (E.sub.1) selected from the CYP152 peroxygenase family, and at least a second genetic mutation that increases the expression relative to the wild type cell of at least one NAD(P)+ oxidoreductase (E.sub.2) and the corresponding mediator protein.

12. The method according to claim 11, wherein E.sub.1 is selected from the group consisting of CYP.sub.SP (E.sub.1a)CYP.sub.BSB (E.sub.1b) and OleT (E.sub.1c): and E.sub.2 and the corresponding mediator protein are selected from the group consisting of ferredoxin reductase (E.sub.2a) and ferredoxin; and putidaredoxin reductase (E.sub.2b) and putidaredoxin.

13. The method according to claim 11, wherein E.sub.1 OleT (E.sub.1c) and comprises at least 60% sequence identity to SEQ ID NO:1; and/or E.sub.2 comprises 60% sequence identity to SEQ ID NO:2 and the mediator protein comprises 60% sequence identity to SEQ ID NO:3.

14. The method according to claim 11, wherein the cell further comprises at least a third genetic mutation that increases the expression relative to the wild type cell of at least one enzyme E.sub.3 capable of NAD(P)H regeneration.

15. The method according to claim 14, wherein the enzyme E.sub.3 is selected from the group consisting of glucose dehydrogenase, phosphite dehydrogenase and formate dehydrogenase.

Description

EXAMPLES

[0075] The foregoing describes preferred embodiments, which, as will be understood by those skilled in the art, may be subject to variations or modifications in design, construction or operation without departing from the scope of the claims. These variations, for instance, are intended to be covered by the scope of the claims.

Example 1

Synthesis of Methionine Starting From Vinylglycine Via Thiol-Ene-Coupling (TEC)

[0076] In a flask (250 mL) is equipped with a reflux condenser vinylglycine (1.011 g, 10.00 mmol, 1.00 eq.) is dissolved in Methanol/Water (1/1, 40 mL) and AIBN (0.164 g, 1.00 mmol, 0.10 eq.) is added. Methyl mercaptan (2.887 g, 2.60 mL, 60.00 mmol, 6.00 eq.) is condensed at 30 C. in a second flask acting as a reservoir. The cooling bath is removed and the reservoir connected to the reaction apparatus to pass the methyl mercaptan through the reaction mixture, while the mixture is heated at 60 C. for 6 hours. The reaction is cooled down to ambient temperature and the formed precipitate collected by filtration to obtain the title compound (as a white crystalline solid of methionine). The structural integrity of the product is confirmed by NMR.

Example 2

Synthesis of Methionine Starting From Vinylglycine Via Thiol-Ene-Coupling (TEC) Under Ambient Pressure

[0077] In a flask (100 mL) equipped with a reflux condenser vinylglycine (1.011 g, 10.00 mmol, 1.00 eq.) is dissolved in methanol/water (1/1, 40 mL) and AIBN (0.082 g, 0.50 mmol, 0.05 eq.) was added. Sodium thiomethoxide (6.205 g, 60.00 mmol, 6.00 eq.) was placed in a second flask and dissolved in distilled water (10 mL). The second flask (50 mL) was equipped with a dropping funnel (25 mL), which contained hydrochloric acid (6 M, 12 ml). The acid was added dropwise to the thiomethoxide solution over a period of 20 minutes to liberate gaseous methylmercaptan, which was passed into the flask with the vinylglycine. The flask with the vinylglycine solution was kept at 60 C. for 12 h. This flask was connected to gas washing bottles, which contained a sodium hydrogen peroxide solution (dist. water (100 mL), H.sub.2O.sub.2 (35%, 40 mL), NaOH (5.21 g)) in order to destroy escaping methylmercaptan. After heating for 12 h, a nitrogen stream was passed through the reaction mixture for 16 h to push all remaining methylmercaptan into the hydrogen peroxide trap. The residual reaction mixture was evaporated and the off-white residue was analyzed by .sup.1H-NMR. The NMR measurement revealed that 1% of the vinylglycine was converted to methionine.

Example 3

[0078] Synthesis of Methionine Starting From Vinylglycine Via Thiol-Ene-Coupling (TEC) Under Excess Pressure

[0079] Vinylglycine (1.011 g, 10.00 mmol, 1.00 eq.) and AIBN (0.082 g, 0.50 mmol, 0.05 eq.) was dissolved in methanol/water (1/1, 40 mL) in a stainless steel autoclave (300 mL). On one side the autoclave was connected to a methylmercaptan gas cylinder via a U-shaped glass tube. The glass tube acted as an intermediate reservoir for methylmercaptan. On the other side the autoclave was connected to gas washing bottles, which contained a sodium hydrogen peroxide solution (dist. water (100 mL), H.sub.2O.sub.2 (35%, 40 mL), NaOH (5.21 g)) in order to destroy escaping methylmercaptan. The whole apparatus was gently flushed with nitrogen for 20 min. Later, the valves of the autoclave were closed and the glass tube was cooled down below 30 C. The gas cylinder was slowly opened to begin condensing of methylmercaptan inside the glass tube. Having condensed a sufficient amount of methylmercaptan (3 mL, 60 mmol, 6 eq.) the gas cylinder was closed again. Next, the autoclave was cooled to below 30 C. and the valve between autoclave and glass tube was opened. The cooling bath of the glass tube was replaced by a water bath to enable condensation of the methylmercaptan inside the autoclave. After complete evaporation of the methylmercaptan inside the glass tube, the autoclave was sealed and the reaction mixture was heated at 60 C. for 18 h (final pressure at 3.5 bar). The autoclave was then cooled down below 30 C. (no excess pressure) and the apparatus was pressurized with nitrogen (ca. 1.2 bar). The valves of the autoclave were carefully opened and a nitrogen stream was passed through the reaction mixture for 22 h to push all remaining methylmercaptan into the hydrogen peroxide trap.

[0080] Cooling of the autoclave after heating sometimes caused a low-pressure in the vessel. In such low-pressure, when the autoclave was opened to the peroxide trap and the applied nitrogen pressure on the other side was not high enough, the peroxide solution was sucked in. Therefore, to prevent this, the vessel was frozen, nitrogen pressure applied and the valve opened between autoclave and glass tube and then the valve between autoclave and trap carefully opened. The nitrogen pressure was increased to establish a stable nitrogen stream through the apparatus when the solution started to get sucked in. increase.

[0081] Then the autoclave was opened and the yellowish residue was suspended in methanol/water (1/1, 40 mL). The precipitated methionine was filtered off, washed with methanol (220 mL) and dried in vacuum. When the condensation of the methylmercaptan proceeded very slowly and the internal pressure of the glass tube raised to 0.6 bar excess pressure, the autoclave valves were opened for some seconds and then later closed. The internal atmosphere was enriched with methylmercaptan and the condensation was faster and proceeded at lower pressures (0.2-0.4 excess pressure).

[0082] Fresh peroxide solution was added in the gas washing bottles before starting the final elimination of methylmercaptan.

[0083] The methionine (0.50 g, 34%, purity.sub.(NMR): 98%) was obtained as an off-white solid. The combined filtrates were evaporated and the residue (0.90 g) was analyzed by .sup.1H-NMR. The NMR measurement revealed that the residue is a mixture of vinylglycine and methionine in a ratio of 27 to 73. Therefore, the overall conversion from vinylglycine to methionine can be calculated to 81%.

Example 4

Synthesis of Methionine Starting From Vinylglycine Via Thiol-Ene-Coupling (TEC) Under Excess Pressure at Lower pH

[0084] Vinylglycine (1.011 g, 10.00 mmol, 1.00 eq.) and AIBN (0.082 g, 0.50 mmol, 0.05 eq.) was dissolved in methanol/water (1/1, 40 mL) in a stainless steel autoclave (300 mL). Acetic acid (1.201 g, 20.00 mmol, 2.00 eq.) was added to the solution (pH=2.5-3). Later, methylmercaptan (3 mL, 60 mmol, 6 eq.) was condensed into the reaction mixture and the autoclave was sealed. The procedure as disclosed in Example 3 was used to handle methylmercaptan. The reaction mixture was heated at 68 C. for 23 h (final pressure at 2.9 bar). The reaction was cooled down and the methylmercaptan was removed. The solvent of the obtained suspension was removed and the residue was washed with EtOH (210 mL). The off-white solid (0.77 g) was dried in vacuum and analyzed by .sup.1H-NMR. The NMR measurement revealed that the residue is a mixture of vinylglycine and methionine in a ratio of 13 to 87.

Example 5

Synthesis of Methionine Starting From Vinylglycine Via Thiol-Ene-Coupling (TEC) Under Excess Pressure Without Radical Starter

[0085] Vinylglycine (1.011 g, 10.00 mmol, 1.00 eq.) was dissolved in methanol/water (1/1, 40 mL) in a stainless steel autoclave (300 mL). Later, methylmercaptan (3 mL, 60 mmol, 6 eq.) was condensed into the reaction mixture and the autoclave was sealed. The procedure as disclosed in Example 3 was used to handle methylmercaptan. The reaction mixture was heated at 66 C. for 22 h (final pressure at 3.2 bar). The reaction was cooled down and the methylmercaptan was removed. The solvent of the obtained solution was removed and the residue was dried in vacuum. Analysis by .sup.1H-NMR revealed that the residue (1.06 g) is a mixture of vinylglycine and methionine in a ratio of 88 to 12. (Conversion rate of vinylglycine to methionine: 12%.)

Example 6

Synthesis of Methionine Starting From Vinylglycine Via Thiol-Ene-Coupling (TEC) Under Excess Pressure With Peroxo Radical Starter

[0086] Vinylglycine (1.011 g, 10.00 mmol, 1.00 eq.) and ammonium peroxodisulfate (0.024 g, 0.10 mmol, 0.01 eq.) was dissolved in methanol/water (1/1, 40 mL) in a stainless steel autoclave (300 mL). Afterwards, methylmercaptan (3 mL, 60 mmol, 6 eq.) was condensed into the reaction mixture and the autoclave was sealed. (For more details how to handle methylmercaptan see procedure above.) The reaction mixture was heated at 66 C. for 23 h (final pressure at 3.2 bar). The reaction was cooled down and the methylmercaptan was removed. The solvent of the obtained solution was removed and the residue was dried in vacuum. Analysis by .sup.1H-NMR revealed that the residue (1.12 g) is a mixture of vinylglycine and methionine in a ratio of 77 to 23. (Conversion rate of vinylglycine to methionine: 23%.)