LU85096A1 - ENZYMATIC SYNTHESIS OF L-SERINE - Google Patents

Description

r 1 • Enzymatic synthesis of L-serine,

The present invention relates to a method for carrying out the synthesis of amino lucid L-serine. The amino acid L-serine is an interesting commercial product which is used in hyperalimentation and nutritional compositions and which is also used as an intermediate product or starting material in certain production processes. synthesis. Therefore, there is a demand for L-serine and various methods have been developed for its preparation. A number of these methods involve producing L-serine by fermentation (see, for example, Morinagà, Y. et al., Agric. Biol. Chem. 45 (6) 1419-24 (198I) \ Morinaga, Y and al., 15 Agric. Biol. Chem. 45 (6) 1425-1430 (198I)). Likewise, in US Patent 3,616,224 granted to the names of Shiio et al., (1971) 3 there is described a process for the production of different amino acids, including serine, by fermentation, process 20 in which strains of certain bacteria are cultivated on media containing methanol as an assimilable carbon source. Reference is also made to United States Patent No. 3 * 943,038 granted to the names of Morinaga et al. (1976) in which a process for the preparation of serine and other amino acids is described by cultivating specific strains of different bacteria in an aqueous culture medium in the presence of oxygen, hydrogen and anhydride carbonic.

A common disadvantage of the fermentation methods described in the prior art is that the concentration of L-serine produced in the broth and recovered is relatively low, even after relatively long fermentations.

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Serine can also be prepared by synthetic chemical processes (see, for example, Kanedo (ed.), ^ Synthetic Production and Utili-zation of Amino Acids11, Halsted Press Books (1974)) · 5 Very often these chemical processes produce the

serine as a racemic mixture of isomers; optical res D and L or in the form of the D isomer

less preferred. Mixtures of DL isomers must be split by methods in which a D-serine catabolizing bacterium, namely serine racemase, by L-amino acid acylase, by fractional crystallization of serine derivatives or by analogous processes, which increases the cost price of the product.

It is known that L-serine can be produced from glycine. The biological production of L-serine from glycine has been carried out with different microorganisms (see, for example, Tanaka, Y. et al., 11 J. Ferment. Technol.11 59: 447 20 (198I) ). As with fermentation processes, a drawback of this process is that the yield of L-serine is not specifically very high.

Therefore, it is necessary to find a method for carrying out the synthesis of L-serine v in high yields. Accordingly, an object of the present invention is to develop a process for the synthesis of L-serine, a process in which the serine is produced in high concentrations in a solution from which it can be efficiently recovered.

Similarly, an object of the invention is to provide an enzymatic means in order to produce L-serine, the reaction being able to be carried out in a continuous or immobilized system.

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It has been found that L-serine can be efficiently synthesized from glycine and formaldehyde in the presence of biocatalysts of the enzyme serine hydroxymethyltransferase and the co-factor tetrahydrofolate under conditions which give rise to serine production. .

* The enzyme can be present in whole cells, in the form of a crude extract or in the form of a purified enzyme and it can be in immobilized form or not.

It is known that the enzyme serine hydroxymethyltransferase catalyzes the cleavage of serine to glycine in a reaction dependent on the co-factors pyridoxal-5’-phosphate and tetrahydrofolate. The reaction gives glycine and methylene tetrahydrofolate. (See Schirck L., Advances in Enzymology 53.:83 (1982)), Now it has been discovered that the enzyme serine hydroxymethyltransferase can be used effectively as a biocatalyst to produce L-serine from glycine and formaldehyde. The reaction takes place in the presence of the cofactor tetrahydrofolate. The source of the tetrahydrofolate may be the source of the serine hydroxymethyltransferase, since the tetrahydrofolate is found in cells of microorganisms containing serine hydroxymethyltransferase or the tetrahydrofolate may be added from an exogenous source. An advantage of this process is that it produces only the 30 L-serine optical isomer. Another advantage is that after carrying out the reaction, the product leaving the reactor contains only glycine and L-serine rather than a complex mixture which one would expect to find in a broth of fermen-35. or following a chemical synthesis.

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It is surprising to find that L-serine can be synthesized from glycine and formaldehyde, since it is known that formaldehyde reacts chemically with proteins and causes the inactivation of enzymes (French, D. et al., “ Advances in Protein Chemistry ”2: 277-335 (1945))" In fact, serine hydroxymethyltransferase is rapidly inactivated by formaldehyde. However, it has been discovered that enzyme 10 can be protected by adding an excess tetrahydrofolate to the reaction mixture, tetrahydrofolate reacts with formaldehyde, protecting the enzyme.

A chemical modification of serine hydroxymethyltransferase also provides stability and protection against inactivation by formaldehyde. For example, it has been found that the reaction of imido-esters with amino groups (see Means, G., et al, Chemical Modification of Proteins, Holden-Day, Inc., 1971) on the surface of the enzyme allowed the enzyme to function in the presence of formaldehyde in higher concentrations than in the case of the unmodified enzyme.

The following is the enzymatic route which is thought to be responsible for the synthesis of L-serine from glycine: formaldehyde + tetrahydrofolate tetrahydro- <5 - methylene folate methylene tetrahydrofolate + 30 glycine '^ ZJIZZZZZZZZ- ZZZZZZZZJIJIZ___I_I___ L- serine + tetrahydrofolate.

It is preferable to carry out the reaction under anaerobic conditions, for example, under a nitrogen atmosphere, in order to prevent oxidation of the tetrahydrofolate.

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The substrate, namely serine hydroxymethyl transferase, as well as the co-factor, namely tetrahydrofolate, can react together in various ways. Although the order in which the reactants and catalysts are introduced is not critical, it is preferable to add glycine and formaldehyde slowly! with serine hydroxymethyltransferase in the presence of tetrahydrofolate. The reaction is carried out under conditions giving rise to the production of L-serine. When the E. coli serum hydroxymethyltransferase (glyA) gene is used as source of serine hydroxymethyltransferase, these conditions generally include a reaction temperature of about 4 to about 60 ° C and a pH in the range of about 4 to about 11. The preferred reaction conditions are to carry out the reaction at a temperature of about 20 to about 45 ° C. and a pH of about 6 to 8.5. If the temperature is less than about 4 ° C., the reaction time is considerably slowed down and, if the The temperature rises above about 60 ° C., the enzyme can be denatured. Similarly, at a pH below about 4 or above about 11, the enzyme can be inactivated. serine hydroxymethyltransferase is a central enzyme in metabolism microorganisms and higher organisms; therefore, there are many virtual sources of this enzyme. The intervals of the conditions in which the L-serine production reaction is carried out are related to the source of the enzyme used. For example, enzymes obtained from thermophilic microorganisms could be used at a temperature higher than that which could be envisaged when the source of

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6 1 * enzyme is E. coli.

The serine hydroxymethyltransferase may be in the form of whole cells, a crude extract or a purified enzyme. The enzyme may be used in immobilized form or not. The enzyme is used in amounts sufficient to catalyze the reaction.

The enzyme can be obtained from microorganisms which have been modified by adopting conventional genetic techniques to produce it with high yields (see Stauffer, G · et al.,

Gene 15: 63-72 (1981)). The serine hydr oxymethyltansferase (glyA) gene can be isolated and cloned into a plasmid which can then be used to transform suitable host cells resulting in the expression of hydroxymethyltans feras e of serine at a high level · Mutant microorganisms which have been modified in their methionine metabolism also give rise to an overproduction of serine hydroxy-20-methyltransferase, (See Stauffer, GV, and Brenchley, JE, Genetics. 88a 221 (1978) and Stauffer, GV · and Brenchley, JE, J. Bacteriol. 129, 740 (1977)) · By adopting gene cloning techniques, the enzymatic activity was increased up to 20 times 25 and it can represent more than $ 10 of the protein. soluble in the cell. A strain E, coli (Gxl703) transformed with such a plasmid, specifically a J - derivative of pBR322 in which the glyA gene has been cloned, pGxl22, has been deposited at the "Northern Regional Research 30 Laboratory", Peoria, Illinois, USA under number NRRL B-15215 · A strain Klebsiella aerogenes (Gxl704) transformed with a similar but smaller plasmid with an alteration causing a high number of copies, pGxl39 has been deposited in the "1 American 35 Type Culture Collection", Rockville , Maryland, USA ·, /

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*, 7 under number ATCC 39214 * while a strain Salmonella typhimurium (Gxl682) transformed with pGxl39 has been deposited under number ATCC 39215 · 1 Furthermore, when the gene is removed from a source such as E. coli “It can be modified by random mutagenesis or by site-directed mutagenesis to produce an enzyme with better stability. Alternatively, the gene can be completely synthesized chemically with multiple changes to improve the stability of the enzyme during the process described.

The use of whole cells can provide a source of tetrahydrofolate. Optionally an additional amount of tetrahydrofolate can be added to achieve saturation levels which are dependent on pH and temperature. For example, at a pH of about 7 * 5 and at a reaction temperature of about 37 ° C, in an aqueous solution, tetrahydrofolate 20 can be added to achieve a concentration well above 50 mM. The pH must be adjusted while the tetrahydrofolate dissolves. If serine hydroxy methyltransferase is added as a crude extract or a purified enzyme, an independent source of tetrahydrofolate is required. The amount ‘of tetrahydrofolate which can be added varies with the temperature and soil conditions, as well as with the pH at which the reaction is carried out.

j 30 It was discovered that tetrahydrofolate j retained its activity with respect to serine hydroxymethyltransferase upon immobilization.

It is advantageous to immobilize the tetrahydrofolate by fixing it on a support which can be retained inside a bioreactor used to carry out the reaction, since this promotes the repeated use of the co- factor at the end of the synthesis of L-serine. For example, tetrahydrofolate can "be immobilized with soluble polymers such as dextran, polyethylene glycol or polyethylene imine. Immobilization takes place by covalent fixation in the first two cases and by ionic interaction in the third case, covalent attachment generally takes place through the intervention of the carboxy groups of the tetrahydrofolate with an amino group of the carrier, or alternatively, by adopting analogous attachment methods, the tetrahydrofolate can also be attached to an insoluble carrier.

Alternatively, if the serine is synthesized in a batch process, the tetrahydrofolate can be recycled. For example, after performing the synthesis of L-serine, the reaction solution can be passed through activated charcoal or through an ion exchange column which retains-20 dra and separates the tetrahydrofolate from the L-solution serine obtained. The tetrahydrofolate can then be released and neutralized. Alternatively, tetrahydrofolate can be modified by covalent attachment to a small molecule such as a glucosamine, to facilitate recovery of the co-factor.

, After carrying out the synthesis of L-serine, the solution of the reactor is passed through a column of borate. Borate binds only to tetrahydrofolate / glucosamine and the modified tetrahydro-folate can be released and recycled.

Preferably, glycine and formaldehyde are added to the mixture of tetrahydrofolate and serine hy-droxymethyltransferase. The amount of glycine which can be added varies with the reaction conditions relating to pH, temperature and solvent, but it can generally be added until the level is reached. saturation is reached.

- As previously indicated, formaldehyde can be highly toxic to serine hydroxymethyltransferase. Consequently, . formaldehyde is usually added slowly to the other components and its addition is controlled. Formaldehyde is added in an amount sufficient to maintain the enzymatic activity, generally to maintain a concentration less than about 10 mM higher than the concentration used for tetrahydrofolate, although formaldehyde can be added to maintain a concentration as high as a concentration about 50 mM higher than the concentration used for tetrahydrofolate. The higher the concentration of tetrahydrofolate in the system, the higher the concentration of formaldehyde can be maintained, since tetrahydro-folate reacts favorably with formaldehyde, thus protecting the enzyme, the reaction mechanism of tetrahydrofolate and formaldehyde is described by Kallen, RG et al., J. Biol. Chem. 241 (24) 585I-5863 (1966). The higher the activity of the enzyme in reactor 25, the faster the formaldehyde is added to maintain the desired concentration.

It has also been found that it may be advantageous to add a second co-factor of serine hydroxymethyltransferase, namely pyridoxal-5 * -phosphate, to the reagents. Pyridoxal-5’-phosphate is firmly bound to the enzyme. If L-serine is synthesized in a reactor over an extended period, the pyridoxal-phosphate may be lost or inactivated, in which case an additional amount of pyridoxal-phosphate may be added. The loss of f,

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* 10 co-factor during the synthesis is indicated by the loss of yellow color of the reaction solution and the loss of activity, by 1 * serum hydroxymethyltrans-ferase. The concentration of pyridoxal-5 phosphate added to the reaction can vary from 0 to about 20 µm (as necessary) and is preferably between about 0.1 µm and about 1 µM.

Adding an excess of pyridoxal-phosphate may perform an additional function. It has been found that in certain microorganisms which have been transformed by plasmids containing the glyA gene and which express high levels of serine hydroxymethyltransferase, the addition of pyridoxal-5'-phosphate is necessary to saturate the hydroxymethyltrans - 15 serine ferase present and improve the enzymatic activity observed. One such microorganism is Klebsiella aerogenes containing the plasmid pGx139 identified above.

The synthesis reaction can be carried out in the presence of any non-harmful solvent.

Among these solvents are, for example, ethanol, inethanol, isopropanol and dioxane.

The following examples are given to illustrate the invention in more detail.

25 Example 1. Bacterial strains were cultivated with and without the plasmid pGxl39 in LB medium (10 g / l • tryptone Bacto, 5 g / l yeast extract, 5 g / l NaCl) or a minimum medium (10, 5 g / l of K ^ HPO ^, 30 4.5 g / l of KH2PO4, 1 g / l of (NH4) 2SO4 and 0.5 g / l of sodium citrate, 21 ^ 0) supplemented with 0.4 $ of glucose or lactose. Where indicated, amino acids were added at a rate of 20 µg / ml. and vitamins at a rate of 1 ^ ig / ml. The specific activity of serine hydroxymethyltransferase is expressed in

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11 nmoles of β-phenyl-serine transformed into benzaldehyde and glycine per minute and per mg of extractable protein after treatment of the cells with ultrasound · 5 The titrations were carried out in a 50 mM phosphate tam pon at a pH of 7 d with 0.1 mM pyri-doxal-phosphate and 35 mM β-phenyl-serine at 20 ° C. The appearance of benzaldehyde was monitored at 279 nM in a recording spectrophotometer.

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Specific activity of serine hydroxymethyltransferase (nmoles / min / mg)

Plasmid strain Medium LB Medium Supplement - minimum 5 Eb coli GX1698 - 39 N.D. * GX1671 pGxl39 221 N.D.

GX1703 pGxl39 141,533 Glucose, ’phenylalanine, thia- mine GX1703 pGxl22 218,682 glucose, 'phenylalanine, thia- mine

Salmonella typhimurium LT2 GX1682 - 28 10 glucose, tryptophan 17 lactose, 20 tryptophan GX1682 pGxl39 152. 133 glucose, tryptophan 306 lactose, tryptophan 25 Klebsiella aerogenes GX1704 - 36 N.D.

GX1704 pGxl39 590 114 glucose 223 108 lactose “P w

Genotype 30 E strain »coli GXI698 trpEA, tna2, serB, laclq ts 402 GX1671 thi, ara j strR, glyA, fserB trpR], laclql, lacZt: tn5

GX1703 glyA, pheA, thi, lac, ara, strR

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Salmonella typhimurium LT2 * GX1682 trpBEDC43 F * laclq ts420 pro

Klebsiella aerogenes? GX1704 lsd (L-serine deaminase mutant).

5 * Not determined.

Strain E, coli GXI67I, which contains the plasmid pGx139, is an additional representative strain which has been used as a source of serine hydro- xymethyltransferase in the process of the present invention.

Example 2

A colony of the Escherichia coli GX 1703 strain (containing pGx122) which was deposited under the number NRRL B-15215 was inoculated into 100 ml of culture medium I described below and stirred at 37 ° C for a night.

Culture medium I: k2hpo4 10.5 g / 1 KH2PO4 4.5 g / 1 20 (NH4) 2SO4 1 g / 1 day Sodium citrate. 2H20 „0.5 g / l

Phenylalanine 20 ^ ig / ml

Vitamin B ^ 1 ^ ig / ml

Ampicillin 100 ^ ig / ml

25 - MgS04 2 mM

'FeSO. 5 mg / ml

Cells were collected by centrifugation from the cell culture medium and used as an enzyme source. Under a nitrogen blanket, glycine (final concentration: 12 mM) was mixed with tetrahydrofolate (final concentration 5 mM), pyridoxal phosphate (1 mM) and formaldehyde (final concentration: 10 mM) · The pH was kept at 7.6 with 0.1 M potassium phosphate buffer and the final volume of this mixture /

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The reaction was 100 ml. The reaction was started by mixing the cells and the above reaction solution at 37 ° C with stirring. After 8 hours, a concentration of 5 mM of serine was obtained (the yield was 45% calculated on the glycine). All the enzymatic activity was retained.

The glycine and serine concentrations were determined by high yield liquid chromatography.

Example 3 A colony of the strain was inoculated

Klebsiella aerogenes GX1704 (containing pGx139) which was deposited under number ATCC 39214j in 100 ml of culture medium XX described below and stirred at 30 ° C overnight.

15 Culture medium II

Trypton e 10 g / 1

NaCl 10 g / 1

Yeast extract 5 g / l

Glucose 10 g / l The cells were collected by centrifugation of the cell culture medium and used as an enzyme source.

The procedure of Example 1 was followed for the production of serine, with the exception that, instead of E. coli, Klebsiella aerogenes was used.

After 8 hours, a concentration of 7 µl of serine was obtained (the yield was 64 ^ 5 calculated on the glycine). All the enzymatic activity was retained.

Example 4 The procedure of Example 1 was followed, with the exception that partially purified serine hydroxymethyl transferase from E. coli (strain GX1703) was used. Cells were broken by treatment with ultrasound at 4 ° C. The supernatant collected after centrifugation was mixed with ammonium sulphate (saturation at $ 50) at 4 ° C and pH 7 * 5. After removal of the solid by centrifugation , the enzyme was removed from the solution bringing the ammonium sulfate content to a saturation of 100%. The enzyme was collected by centrifugation and subjected to dialysis,. After reaction for 4 hours, a concentration of 10 μm of serine was obtained. The yield was $ 90, Galculé sur la glycine. All enzyme activity was retained.

Example 5

The procedure of Example 3j was followed with the exception that the initial glycine concentration was 340 mM and that the form% maldehyde (initial concentration: 2 m) was introduced at a rate of 1 ml / hour. After 12 hours, a concentration of 119 mM of serine was obtained.

Example 6

The procedure of Example 3 was followed, with the exception that glycine (initial concentration 2 2M) and formaldehyde (initial concentration 2 2M) were introduced at the same rate of 1 ml / hour. After 5 hours, a concentration of 57 mM of serine was obtained.

25 'Example 7

The procedure of Example 3j was followed with the exception that the tetrahydrofolate was modified. 5 g of dextran ("Pharmacia T40") were dissolved in water to a final concentration of 5% · The dextran solution was oxidized 30 · using 0.1 M NalO 4 for 1 hour at room temperature. The oxidized dextran was precipitated by the addition of ethanol to $ 60 (volume / volume). This step was repeated twice.

The oxidized dextran was dissolved in 100 ml of 1.6-35 0.2M hexane diamine at a pH of 9 · 0.2 g was added

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"16 sodium borohydride after 30 and 60 minutes respectively. The 1,6-hexane diamine / dextran was dialyzed against water overnight and lyophilized. Tetrahydrofolate (5 mM) was mixed with 10 µM 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide and 0.5% of. 1,6-hexane-diamine / dextran at a pH of 7 under a nitrogen atmosphere. The precipitate formed was collected by centrifugation and washed twice with 0.5M NaCl solution. The solid tetrahydrofolate was mixed with the reaction mixture as described in Example 3 · After reaction for 3 hours, a concentration of 7 "M of serine was obtained.

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Claims (25)

4 17 1. Process for the synthesis of L-serine, characterized in that it consists in reacting glycine and formaldehyde in the presence of biocatalytic quantities of serine hydroxymethyltransferase and tetrahydrofolate under conditions of production of L-serine. 2. Method according to claim 1, characterized in that the temperature is maintained between 1Ό about 4 eh about 60 ° C, while the pH is in the range from about 4 to about 11. 3. Method according to claim 2, characterized in that the temperature is in the range from about 20 to about 45 ° C, while the pH is in the range from about 6 to about 8.5. 4 »Process according to claim 1, characterized in that the serine hydroxymethyltransferase is contained in whole cells. 5. The process according to claim 1, ca characterized in that the serine hydroxymethyltransferase is in the form of a crude extract. 6. Method according to claim 1, characterized in that the serine hydroxymethyltransferase is in the form of a purified enzyme. 7. Method according to claim 1, characterized in that the serine hydroxymethyltransferase is immobilized. 8. Process according to claim 1, characterized in that the reaction is carried out in the form of a batch system. 9. Method according to claim 1, characterized in that the reaction is carried out in the form of a continuous system. L ~ 9 18 10. Process according to claim 1, characterized in that the source of tetrahydrofolate consists of whole microbial cells - containing the serine hydroxymethyltransferase. 11. Method according to claim 10, characterized in that an additional quantity of tetrahydrofolate is added to the reaction system in order to bring the concentration of tetrahydrofolate (* to a maximum of the degree of saturation. 12. The method of claim 1, characterized in that the concentration of tetrahydrofolate is in the range of from about 0.15 to about 50 mM. 13 · A method according to claim 1,% 15 characterized in that the tetrahydrofolate is immobilized. 14. Method according to claim 13, characterized in that the tetrahydrofolate is immobilized with a soluble polymer, 15. The method of claim 133 characterized in that the tetrahydrofolate is immobilized by attachment to a solid support. 16. The method of claim 1, characterized in that the tetrahydrofolate is modified so that it can be recycled. 17 · Process according to claim 1, characterized in that the glycine can be added: - until the reaction solution is saturated with it. ; , 30 A method according to claim 1, characterized in that formaldehyde is added to a maximum concentration higher from about 30 mM to about 50 mM than the concentration of tetrahydrofolate. L 19 19 · A method according to claim 18, characterized in that the concentration of formaldehyde is brought to a constant state of a concentration— "tion greater than about 10 mM to the concentration 5 of tetrahydrofolate, 20. The method of claim 1, characterized in that pyridoxal-phosphate is added to the reagents at a concentration of 0 to about 20 mM. 21. The method of claim 20, characterized in that the concentration of pyridoxalphosphate is in the range of about 0.1 to 1.0 mM. 22. The method of claim 1, characterized in that the source of serine hydroxymethyltransferase is the strain Escherichia coli GX1703 containing the plasmid pGxl22 and deposited at the "Northern Regional Research Laboratory" under number NRRL B-15215. 23. The method of claim 1, characterized in that the source of serine hydroxymethyltrans-ferase is Marigold Salmonella thyphimurium Gxl682, containing the plasmid pGxl39 and deposited in the "American Type Culture Collection" under number ATCC 39215. 24. The method of claim 1, characterized in that the source of serine hydroxymethyltrans ferase is the strain Klebsiella aerogenes GX1704j containing the plasmid pGxl39 and deposited in the "American Type Culture Collection" under number 30 ATCC 39214. 25. The method of claim 1, characterized in that the activity of serine hydroxymethyl transferase in cells has been amplified by genetic manipulation. h ‘i 20 26, Method according to claim 1,! characterized in that the serum hydroxymethyltransferase activity in cells has been amplified by cloning the hydroxymethyltransferase gene from | 5 serine into a plasmid and by transformation of a host cell with this plasmid to give rise to; j - an overproduction of hydroxymethyltransferase from; i serine. k; 27 · Process according to claim 1, i 10 characterized in that the hydroxymethyltransferase of i serine is obtained from any i biological source. i 28, A method according to claim 1, characterized in that the serine hydroxymethyltrans-15 ferase gene has been modified by random mutagenesis or by site-directed mutagenesis to increase the stability of the enzyme, 29. The method of claim 1, characterized in that the enzyme serum hydroxymethyltransferase is chemically modified for ac-1! increase its stability. 30. The method according to claim 29, characterized in that the enzyme is modified by reaction with imido-esters, pratiquement 25 31 · An essentially biologically pure culture of the Escherichia coli GX1703 strain containing 1 the plasmid pGx122, îj: j ~ 32. Virtually biologically r> i: pure culture of the strain Salmonella typhimurium GX1682 containing the plasmid pGxl39 ·, | 33 · Virtually biologically pure culture of the strain Klebsiella aerogenes ^ GX1704 cont- H =! λ ning the plasmid of pGxl39 · i