FR2536415A1 - PROCESS FOR THE ENZYMATIC SYNTHESIS OF L-SERINE - Google Patents

Abstract

PROCESS FOR THE SYNTHESIS OF L-SERINE FROM GLYCINE AND FORMALDEHYDE IN THE PRESENCE OF BIOCATALYTIC QUANTITIES OF HYDROXYMETHYLTRANSFERASE AND TETRAHYDROFOLATE.

Description

25364 1 5

  Process for the enzymatic synthesis of L-serine The present invention relates to a process for the synthesis of the amino acid called L-serine This amino acid is a valuable commercial product which is used in hyperalimentation and in nutritional compositions and which is also used as an intermediate or as a starting material in certain synthetic methods There is therefore a demand for L-serine, and various methods have been developed for producing it A certain number of these methods

  involve the production of L-serine by fermentation.

  See, for example, Morinaga, Y, et al, Agric Biol Chem. (6) 1419-24 (1981; Morinaga, Y, et al, Agric Biol Chem. (6) 1425-1430 (1981) On the other hand, U.S. Patent No. 3,616,224 describes a process allowing the production of various amino acids, including serine, by fermentation, in which strains of certain bacteria are cultivated on media containing methanol as source

  of assimilable carbon See also the United States patent

  United States of America N 3 943 038, which describes a process for the preparation of serine and other amino acids by culturing specific strains of various bacteria in an aqueous culture medium in the presence of oxygen, hydrogen and gas carbonic. A common drawback to the fermentation processes taught by the prior art is that the concentration of L-serine produced in the broth and recovered is relatively low, even after

relatively long fermentations.

  Serine can also be produced by chemical synthesis processes See for example Kanedo (ed), Synthetic Production and Utilization of Amino Acids, Halsted Press Books (1974) Often these chemical processes produce serine as a racemic mixture optical isomers D and L or in the form of the isomer D which is the least interesting DL mixtures must be resolved by

  processes that use bacteria catabolizing D-

  serine, the serine racemase, by amino acid acylase L, by fractional crystallization of serine derivatives, or by similar methods, which adds to the cost price of the product. We know that Lserine can be produced from glycine Biological production of L-serine from

  glycine has been accomplished with several microorganisms.

  See for example Tanaka, Y, et al, J Ferment Technol, 59: 447 (1981) As with fermentation processes, a

  disadvantage of this method is that the yield of L-

  serine is generally not very high.

  There is therefore a need for a process for the synthesis of L-serine with a high yield. It is therefore an objective of the present invention to develop a process for the synthesis of L-serine in which the serine is produced with a high concentration in a solution to

  from which we can recover it effectively.

  It is also an object of the present invention to provide an enzymatic means for the production of L-serine, in which the reaction can be carried out batchwise or

in an immobilized system.

  It has been discovered that L-serine can be efficiently synthesized from glycine and formaldehyde in the presence of biocatalytic amounts of a

  enzyme, serine hydroxymethyltransferase, and a co-

  factor, tetrahydrofolate, under conditions suitable for producing serine The enzyme can be found in whole cells, in the form of a crude extract, or in the form of a purified enzyme and can be in a

  immobilized or non-immobilized form.

  It is known that serine hydroxymethyltransferase (hereinafter SHMT) is an enzyme which catalyzes the cleavage of the

  serine to glycine in a reaction which depends on two co-

  factors, pyridoxal 5 'phosphate and tetrahydrofolate.

  The reaction gives glycine and methylene tetrahydro-

  folate See Schirck, L, Advances in Enzymology 53:83 (1982) It has now been discovered that the enzyme SHMT / can be used effectively as a biocatalyst for

  produce L-serine from glycine and formaldehyde.

  The reaction takes place in the presence of the co-factor tetrahydrofolate (THF) The source of THF may be the source of SHMT, because THF is found in the cells of microorganisms which contain SHMT, or else THF may be added from an exogenous source One of the advantages of this process is that it produces only the optical L isomer of serine A

  another advantage is that, after the reaction has taken place, -

  the reactor product contains only glycine and L-serine, not a complex mixture such as that one would expect to find in a fermentation broth or

following a chemical synthesis.

  It is surprising that L-serine can be synthesized from glycine and formaldehyde, because we know 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, SHMT is quickly inactivated by formal-

  dehyde But it was discovered that the enzyme could be protected by adding an excess of tetrahydrofolate to the reaction mixture. THF reacts with formaldehyde,

thus protecting the enzyme.

  The chemical modification of the SHMT also ensures

  its stability and protects it from inactivation by formal-

  dehyde As an example, it has been found that the reaction of imidoesters with amine groups (see Means, G, et al., Chemical Modification of Proteins, Holden-Day, Inc, 1971) on the surface of the enzyme allows the latter to function in the presence of higher concentrations of the

  formaldehyde as the unmodified enzyme.

  The enzymatic path that is thought to explain the synthesis of L-serine from glycine is as follows: formaldehyde + THF <-> methyleneTHF mthyl Sne-THF + glycineL-srine + THF methylene-THF + glycine <Lserine + THF It is preferable that the reaction is carried out under anaerobic conditions, for example in an atmosphere

  nitrogen, to prevent oxidation of THF.

  Substrates, SHMT and co-factor THF can be reacted together in different ways Although the order in which the reactants and catalysts are introduced is not critical, it is preferable that glycine and formaldehyde be added slowly at the SHMT in the presence of THF The reaction is carried out

  under conditions suitable for producing L-serine.

  When the E coli SHMT gene (gly A) is used as the source of SHMT, these conditions generally include a reaction temperature of about 4 to about 600 C and a p H in the range of about 4 to about The preferred reaction conditions include a temperature of about 20 to about 450 C and a p H of about 6 to 8.5 If the temperature is less than about 40 C, the reaction time is

  considerably elongated, and if the temperature rises

  above 60 ° C the enzyme may be denatured Likewise, at a p H less than 4 or greater than 11 approximately, the enzyme may be inactivated SHMT is a central enzyme in the metabolism of microorganisms and higher bodies; there are also many potential sources of the enzyme.

  in which the L-serine production reaction is.

  carried out are linked to the source of the enzyme used. For example, enzymes derived from thermophilic microorganisms could be used at a higher temperature than is possible when the source of the enzyme

is E coli.

  The serine hydroxymethyltransferase can optionally be in the form of whole cells, of a crude extract, or of a purified enzyme The enzyme can be

  used in immobilized or non-immobilized form.

  The enzyme is used in sufficient amounts to catalyze

the reaction -

  The enzyme can be derived from microorganisms which have been modified using conventional genetic manipulation techniques to produce it with high yields See Stauffer, G, et al, Gene 15: 63-72 (1981) isolate the SHMT gene (gly A) and clone it into a plasmid, which can then be used to transform the appropriate host cells resulting in a high level of SHMT expression. The mutant microorganisms which have been modified in their methionine metabolism also overproduces SHMT See Stauffer, GV, and Brenchley, JE, Genetics 88, 221 (1978) and Stauffer, GV, and Brenchley, JE, J. Bacteriol 129, 740 (1977) Using cloning techniques of the genes, the enzymatic activity has been increased to multiply it by twenty, which can represent more than 10% of the soluble proteins of the cell A strain of E coli (Gx 1703) transformed by such a plasmid, more precisely a derived from p BR 322 in which the gly A gene was cloned, p Gx 122, was deposited at the Northern Regional Research Laboratory of Peoria, Illinois under the number NRRL B-15215 A strain of Klebsiella aerogenes (Gx 1704) transformed by a similar but smaller plasmid, with an alteration causing a large number of replication, p Gx 139, was deposited in the American Type Culture Collection of Rockville, Maryland under the n ATCC 39214, and a strain of Salmonella typhimurium (Gx 1682) transformed by p Gx 139

  was filed under N ATCC 39215.

  In addition, when the gene comes from a source

  such as E coli, it can be modified by random mutagenesis -

  roofing or site-directed mutagenesis to produce an enzyme with greater stability Or, the gene can be obtained by total chemical synthesis with multiple modifications to improve the stability of

  the enzyme during the process described.

  The use of whole cells can constitute a source of THF If necessary, a supplement of THF can be added to reach saturation levels, which depend on p H and temperature. For example, at p H of About 7.5 and at a reaction temperature of about 37 C, in aqueous solution, THF can be added so as to reach a concentration well above 50 m M. The p H must be adjusted while the THF dissolves If the SHMT is added in the form of a raw extract or a

  purified enzyme, an independent source of THF is required.

  The amount of THF which can be added varies with the temperature, the conditions of the solvent and the p H at which the

reaction is conducted.

  It has been discovered that THF retains its activity with respect to SHMT when it is immobilized. It is advantageous to immobilize THF by fixing it on a support which can be stored inside a bioreactor used to carry out the reaction, since this promotes the repeated use of the co-factor after the synthesis of L-serine is completed. For example, THF can be immobilized with soluble polymers, such as dextran,

  polyethylene glycol or polyethyleneimine.

  The immobilization is done by covalent bond in the first two cases and by ionic interaction in the third. The covalent bond is generally done through the THF carboxyl groups with an amine group of the support Or, using bonding modes similar, THF can also be fixed on an insoluble support. Or, if the serine is synthesized according to a discontinuous process, it is possible to recycle the THF For example, after the L-serine has been synthesized, the reaction solution can be passed over active carbon or in a ion exchange column, which will retain and separate the THF from the L-serine solution produced We can then release the THF and neutralize it Or else, we can modify the THF by fixing it by covalent bonding on a small molecule, such as glucosamine, to facilitate recovery of the co-factor After the L-serine has been synthesized, the solution which leaves the reactor is passed over a column of borate The borate binds only to the THF-glucosamine combination, and the modified THF can be

released and recycled.

  Glycine and formalin are preferably added.

  dehyde to the mixture of tetrahydrofolate and SHMT The amount of glycine that can be added varies with p H, the temperature and the conditions of the reaction solvent, but it can generally be added until the level of saturation is reached. As indicated previously, formaldehyde can be very toxic towards SHM To. This is why, generally, formaldehyde is added slowly to the others.

  components, and we regulate its addition We add the formal-

  dehyde in sufficient quantity to maintain the enzymatic activity, generally so as to maintain a concentration less than about 10 m M higher than the concentration of THF used, although it is possible to add formaldehyde so as to maintain a higher concentration of not less than about 50 m M at the concentration of THF used The higher the concentration of THF in the system, the higher the concentration of formaldehyde can be maintained, because THF reacts favorably with formaldehyde, thus protecting the enzyme The mechanism of the reaction of THF and formaldehyde is described by Kallen, RG, et al, J Biol Chem 241 (24) 5851-5863 (1966) The greater the activity of the enzyme in the reactor, the more rapidly the formaldehyde to maintain the

desired concentration.

  -25 It was also discovered that it could be

  advantageous to add a second SHMT co-factor, 5 '-

  pyridoxal phosphate, to reactants Le 5 '-

  pyridoxal phosphate binds tightly to the enzyme If L-serine is synthesized in a reactor over an extended period of time, pyridoxal phosphate may disappear or become inactive, in which case a supplement of pyridoxal phosphate La disappearance of the co-factor during synthesis is indicated by the disappearance of the yellow color from the reaction solution and by the loss of activity of the serine hydroxymethyltransferase The concentration of pyridoxal phosphate added to the reaction mixture can vary from 0 to about 20 m M, as required, and is preferably from about 0.1 m M to about 1 m M.

25364 15

  The addition of excess pyridoxal phosphate may serve an additional function. It has been discovered that in certain microorganisms which have been transformed by plasmids containing the gly A gene and which express high levels of SHMT, the addition of 5 '-pyridoxal phosphate is necessary to saturate the SHMT present and to improve the enzymatic activity observed One of these microorganisms is Klebsiella aerogenes containing the

  plasmid p Gx 139 identified above.

  The synthesis reaction can be carried out in the presence of any non-deleterious solvent. Examples of these solvents include ethanol, methanol, isopropanol and

dioxane.

  The following examples are intended to describe

the invention in more detail.

EXAMPLE 1

  Bacterial strains were cultivated with and without plasmid p Gx 139 in LB medium (10 g / liter of tryptone Bacto, 5 g / liter of yeast extract, 5 g / liter of sodium chloride) or in a medium minimum (10.5 g / liter of K 2 HPO 4, 4.5 g / liter of KH 2 PO 4, 1.0 g / liter of (NH 4) 2 SO 4 and 0.5 g / liter of citrate of sodium bihydrate), supplemented with 0.4% glucose or lactose 20 mg / ml of amino acids and 1 gg / ml of vitamins were added in the cases indicated The specific activity of serine hydroxymethyltransferase is

  expressed in nmoles of f-phenyl serine transformed into benz-

  aldehyde and glycine per minute and per mg of protein

  extractable after sonication of the cells.

  The assays were carried out in 50 m M of phosphate buffer at p H 7.3 with Q, 1 μm of pyridoxal phosphate and m M of g-phenylserine at 20 ° C. The appearance of benzaldehyde was monitored at 279 n M in a recording spectrophotometer. Specific SHMT (nmoles / min / mg) Plasmid p Gx 139 p Gx 139 p Gx 122 Medium LB 39 - Minimum medium ND * ND Supplement glucose, phenylalanine, thiamine glucose, phenylalanine, thiamine Salmonella typhimurium LT 2 GX 1682 28 10 glucose, tryptophan 17 lactose, tryptophan GX 1682 p Gx 139 152 133 glucose, tryptophan 306 lactose, tryptophan Klebsiella aerogenes

GX 1704 36 N D.

  GX 1704 p Gx 139 590 114 glucose 223 108 lactose Strain Genotype E coli GX 1698 trp EA, tna 2, ser B, lac Iq ts 402 GX 1671 thi, ara, str R, gly A, lser B trp Rl, lac Iql, lac Z :: tn 5 GX 1703 gly A, phe A, thi, lac, ara, str R Salmonella typhimurium LT 2 GX 1682 trp BEDC 43, F 'lac Iq ts 420 pro Klebsiella aerogenes GX 1704 lsd (L deaminase mutant - serine) * not determined Strain GX 1671 from E coli, which contains the plasmid p Gx 139, is a representative additional strain which was used as a source of

  SHMT in the process according to the present invention.

E coli strain activity

GX 1698

GX 1671

GX 1703

GX 1703

2536-415

''

EXAMPLE 2

  A colony of strain GX 1703 of Escherichia coli (containing p Gx 122), which was deposited under N NRRL B-15215, was inoculated in 100 ml of culture medium I

  described below, and shaken at 37 ° C overnight.

  Culture medium I: K 2 HPO 4 10.5 g / 1 KH 2 PO 4.5 g / l

2 /

  (NH 4) 2 SO 4 1 g / 1 Sodium citrate 2 H 20 0.5 g / l Phenylalanine 20 gg / ml Vitamin Bl 1 ig / ml Ampicillin 100 _g / ml Mg S 2 m M Me SO 4 Fe SO 4 5 mg / ml The cells were collected by centrifugation of the cell culture medium and used as a source of the enzyme. Glycine (12 m M, final concentration) was mixed with tetrahydrofolate (5 m M, final concentration), pyridoxal phosphate (1 m M) and formaldehyde (10 m M, final concentration) under nitrogen The p H was kept at 7.6 with 0.1 M potassium phosphate buffer, and the final volume of this reaction mixture was ml The reaction was started by mixing the above-mentioned reaction solution and the cells at 37 C by shaking After 8 hours, 5 m M of serine were produced (the yield was 45% compared to glycine) All of the enzyme activity was retained. The concentration of glycine was determined and

  serine by high performance liquid chromatography.

EXAMPLE 3

  A colony of strain GX 1704 from Klebsiella aerogenes (containing p Gx 139), which was deposited under N ATCC 39214, was inoculated into 100 ml of culture medium II

  described below, and shaken at 30 ° C overnight.

  Culture medium II Tryptone 10 g / l Na Cl 10 g / l Yeast extract 5 g / l Glucose 10 g / l The cells were collected by centrifugation of the cell culture medium and used as

source of the enzyme.

  The procedure of Example 2 was followed for the production of serine, except that Klebsiella aerogenes was used in place of Eo coli. After 8 hours, 7 m M of serine were produced ( the yield was 64% compared to glycine) The entire activity

enzymatic has been preserved.

EXAMPLE 4

  The procedure of Example 2 was followed, except that partially purified serine hydroxymethyltransferase from E coli (strain GX 1703) was used. The cells were broken by sonification at 4 Co. mixed the supernatant collected after centrifugation with

  ammonium sulfate (50% saturation) at 4 C and at p H 7.5.

  After separating the solid by centrifugation, the enzyme was expelled from the solution bringing the ammonium sulphate content to 100% of saturation. The enzyme was collected by centrifugation and dialyzed 10 m M from serine were produced after 4 hours of reaction The yield was% compared to glycine All the enzymatic activity

kept.

EXAMPLE 5

  The procedure of Example 3 was followed, except that the initial concentration of glycine was 340 m M and that formaldehyde (initial concentration of 2 M) was introduced at a flow rate of 1 ml / hour After 12

  hours, 119 m M of serine were produced.

EXAMPLE 6

  The procedure of Example 3 was followed, except that glycine (initial concentration of 2 M) and formaldehyde (initial concentration of 2 M) were introduced at the same flow rate of 1 ml per hour At the end 5 hours, 57 m M of

serine have been produced.

EXAMPLE 7

  The procedure of Example 3 was followed, except that the THF was modified. Dextran (5 g, Pharmacia T 40) was dissolved in water to a final concentration of 5%. It was oxidized. the dextran solution with 0.1 M Na IO 4 for one hour at room temperature The oxidized dextran was precipitated by adding 60% ethanol (volume / volume) This operation was repeated twice. dissolved oxidized dextran in 100 ml of 1,6-hexanediamine (HMD) 0.2 M at w H 9.0 Sodium borohydride (0.2 g) was added after 30 and 60 minutes respectively We dialyzed HMD-dextran with water overnight and

  freeze dried THF (5 m M) was mixed with 10 m M of 1-

  ethyl-3- (3-dimethylaminopropyl) carbodiimide and HMD-

  0.5% dextran at p H 7.0 under nitrogen atmosphere The precipitate formed was collected by centrifugation and washed twice with 0.5 M sodium chloride solution. THF was mixed solid with the reaction mixture as described in Example 3 After three hours of reaction, 7 m M

of serine have been produced.

Claims (23)

  1 L-serine synthesis process, characterized   in that it involves reacting glycine and formal-   dehyde in the presence of biocatalytic amounts of serine hydroxymethyltransferase and tetrahydrofolate in   specific conditions for producing L-serine.   2 Method according to claim 1, characterized in that the temperature is maintained between 4 C approximately and 60 C and the p H between 4 approximately and 11 approximately, 3 Method according to Claim 2, characterized in that the temperature is included between about 20 C and    C approximately and the p H between approximately 6 and 8.5 approximately.   4 Method according to claim 1, characterized in that the serine hydroxymethyltransferase is contained in whole cells.   Process according to claim 1, characterized in that the serine hydroxymethyltransferase is under the form of a crude extract.   6 Method according to claim 1, characterized in that the serine hydroxymethyltransferase is under the form of a purified enzyme.   7 Method according to claim 1, characterized   in that the serine hydroxymethyltransferase is immobilized.   8 Method according to claim 1, characterized   in that the reaction is carried out batchwise.   9 Method according to claim 1, characterized   in that the reaction is carried out continuously.   Process according to claim 1, characterized in that the source of tetrahydrofolate consists of   whole microbial cells containing hydroxymethyl- serine transferase.   11 Method according to claim 10, characterized in that an addition of tetrahydrofolate is added to the reaction mixture to bring the concentration of tetrahydrofolate at the maximum of the saturation level.   12 Method according to claim 1, characterized in that the concentration of tetrahydrofolate is included   between about 0.15 m M and about 50 m M.   13 Process in which we immobilize 14 Process in which we immobilize soluble polymer. Process in that one immobilizes solid support. 16 Procedure in that the we can recycle it.   17 Method according to claim 1, characterized tetrahydrofolate.   according to claim 13, characterized tetrahydrofolate using a according to claim 13, characterized tetrahydrofolate by fixing it on according to claim 1, characterized tetrahydrofolate so that according to claim 1, characterized in that one adds glycine until the reaction solution is saturated with glycine 18 Process according to claim'1, characterized in that formaldehyde is added to a higher maximum concentration of approximately 30 m M to approximately 50 m M to the THF concentration.   19 A method according to claim 18, characterized in that the concentration of formaldehyde is brought to a permanent state in which it is greater by about m M at the THF concentration.   20 Method according to claim 1, characterized in that pyridoxal phosphate is added to the products   reactants, at a concentration of O to about 20 m M.   21 Method according to claim 20, characterized in that the concentration of pyridoxal phosphate is between 0.1 and 1.0 m M approximately.   22 Method according to claim 1, characterized in that the source of SHMT is the strain GX 1703 of Escherichia coli, containing the plasmid p Gx 122, deposited at the Northern Regional Research Laboratory under the N NRRL B-15215.   23 Method according to claim 1, characterized in that the source of SHMT is the strain GX 1682 of Salmonella typhimurium, containing the plasmid'p Gx 139, deposited at   the American Type Culture Collection under N ATCC 39215.   24 Method according to claim 1, characterized in that the source of SHMT is the strain GX 1704 of Klebsiella aerogenes, containing the plasmid p Gx 139, deposited in the American Type Culture Collection under the N ATCC 39214. Method according to claim 1 , characterized in that the activity of serine hydroxymethyltransferase in cells has been amplified by genetic manipulation. 26 Method according to claim 1, characterized in that the activity of serine hydroxymethyltransferase in the cells has been amplified by cloning of the gene for serine hydroxymethyltransferase into a plasmid, and transformation of a host cell by this plasmid for   overproduce serine hydroxymethyltransferase.   27 A method according to claim 1, characterized in that the serine hydroxymethyltransferase is derived from a any biological source.   28 Method according to claim 1, characterized.   in that the serine hydroxymethyltransferase gene has been modified by random mutagenesis or by site-directed mutagenesis   on the site so as to increase the stability of the enzyme.   29 Method according to claim 1, characterized in that chemically modifies the hydroxymethyltransferase   of serine so as to increase the stability of the enzyme.   Process according to Claim 29, characterized in that the enzyme is modified by reacting it with imido-esters. 31 A substantially pure culture, from a biological point of view, of GX 1703 strain of Escherichia coli containing the plasmid p Gx 122.   32 A substantially pure culture, from a biological point of view, of strain GX 1682 of Salmonella typhimurium containing the plasmid p Gx 139.   33 A substantially pure culture, from a biological point of view, of strain GX 1704 of Klebsiella aerogenes containing the plasmid p Gx 139.