EP1404854A2

EP1404854A2 - Process for the preparation of enantiomer-enriched amino acids

Info

Publication number: EP1404854A2
Application number: EP02711530A
Authority: EP
Inventors: Wilhelmus Hubertus Joseph Boesten; Joannes Gerardus Theodorus Kierkels
Original assignee: DSM IP Assets BV
Current assignee: DSM IP Assets BV
Priority date: 2001-01-31
Filing date: 2002-01-31
Publication date: 2004-04-07
Also published as: WO2002061107A2; NL1017250C1; KR20030071868A; WO2002061107A3; CN1520460A; CZ20032077A3; AU2002230274A1; JP2004521623A; HUP0302864A2

Abstract

Process for the preparation of a chiral amino acid enriched in the D-enantiomer, in which a mixture of the enantiomers of the corresponding N-carbamoylamino acid is brought into contact with a D-carbamoylase with ammonia being liberated, the ammonia being removed with the aid of a bivalent metal salt of a phospahte, a monohydrogen phosphate or a dihydrogen phosphate ion. In one embodiment the enzymatic decarbamoylation is carried out in the presence of a bivalent metal salt of a phosphate ion, a monohydrogen phosphate ion or a dihydrogen phosphate ion. In another embodiment the reaction mixture is brought into contact via an external loop, after separation of the solid present, with the bivalent metal salt of a phosphate ion, monohydrogen phosphate ion or dihydrogen phosphate ion. The chiral amino acid enriched in the D-enantiomer can also be obtained by enzymatically converting the corresponding hydantoin with the aid of a hydantoinase into the corresponding N-carbamoylamino acid, which is subsequently converted according to the invention into the amino acid enriched in the D-enantiomer.

Description

PROCESS FOR THE PREPARATION OF ENANTIOMER-ENRICHED AMINO ACIDS

The invention relates to a process for the preparation of an amino acid enriched in the D-enantiomer, in which a mixture of the enantiomers of the corresponding N-carbamoylamino acid is brought into contact with a D-carbamoylase with ammonia being liberated. D-amino acids are important building blocks for biologically active preparations such as β-lactam antibiotics, peptide hormones and pesticides. A commonly used preparation process for these "unnatural" amino acids is a process in which the corresponding DL-5-substituted hydantoin is enantioselectively hydrolysed by a hydantoinase to form the corresponding N-carbamoylamino acid. This N- carbamoylamino acid can be converted enzymatically to the corresponding D-amino acid.

A disadvantage of the known process is that, to realise a certain reaction time, relatively large quantities of biocatalyst are necessary because it is generally known that the enzyme that is responsible for the hydrolysis of carbamoylamino acids (the carbamoylase, also called N-carbamoyl-D-amino acid amidohydrolase) is strongly inhibited by the reaction product ammonia. The concentration at which ammonia-caused inhibition occurs is reported (Olivieri et. al. (1981) Biotechnology and Bioengineering, vol. 23, 2173-2183; Runser (1990) Applied Microbiology and Biotechnology, 33, 382-388; Louwrier (1996) Enzyme and Microbial Technology, 19, 562-571 ) to be very low (- 10 mM). For the known processes this means that at an ammonia concentration of 10 mM the carbamoylase is active at half the maximum rate. At an ammonia concentration of 20 mM the rate of the carbamoylase is only 1/3 of the maximum rate. It will be clear that under industrially relevant conditions, where high product concentrations, for example > 500 mM, are desirable, the rate of the carbamoylase will be very strongly inhibited. Lower product concentrations (order of magnitide 10 mM) at which the activity of the carbamoylase is almost maximum are economically unattractive because of its low production capacity. In the literature various processes are described to keep the ammonia concentration low during the enzymatic conversion. An example of in- situ removal of ammonia is described by Kim & Kim, (1995), Enzyme and Microbial Technology, 17, 63-67, where use is made of an adsorbent (AD300NS, a silicate complex of Tomita Pharmaceutical Co. (Japan)), for the removal of ammonia during the preparation of D-p-hydroxyphenylglycine with the aid of hydantoinase and carbamoylase. A disadvantage of this process is that the adsorbent is expensive and thus must be recycled.

According to another process, described in J 61.285.996-A (1986) the ammonia formed is removed by distillation. However, this process for the removal of ammonia is effective only when the reaction mixture has a high pH. At such a high pH, however, the carbamoylase enzyme is not active. When ammonia is removed by distillation at neutral pH, between pH 7-7.5, 60 to 90% of the formed ammonia remains in the reaction mixture even when more than 90% of the reaction volume is removed. Therefore, efficient removal at neutral pH is not possible.

The invention now provides a simple and economically attractive process in which less biocatalyst needs to be used or a shorter reaction time is realised. This is achieved according to the invention by removing the ammonia with the aid of a bivalent metal salt of a phosphate ion, a monohydrogen phosphate ion or a dihydrogen phosphate ion; in the further description briefly designated as phosphate salt. To this end for example the enzymatic reaction can be carried out (for example) in the presence of a phosphate salt. Surprisingly it has furthermore been found that the reaction mixture remained easily stirrable also at high slurry concentrations.

Another embodiment is formed for example by leading the reaction mixture via for example a loop after separation of the undissolved reaction components, through for example a second reactor, or a column or a filter in which the phosphate salt is present. The ammonia present in the reaction mixture is then bound to the phosphate salt, yielding the corresponding ammonium phosphate salt, after which the remaining liquid, which still contains for example enzyme, is returned to the enzymatic decarbamoylation reaction vessel. It has been found that no or much less enzyme inhibition takes place. This is all the more surprising because it is known that divalent metals can interfere with carbamoylase-catalysed reactions even at low concentrations (1 -10 μ molar).

Examples of suitable bivalent metal ions are magnesium, cobalt, calcium, manganese, zirconium or ruthenium ions. For economic reasons use is preferably made of magnesium monohydrogen phosphate (MgHPO₄), also called magnesium hydrophosphate. MgHPO₄fits in with the optimum process conditions as regards the resulting pH and is easy to prepare from cheap raw materials. The phosphate salt can also be formed in situ.

The phosphate salt, for example MgHPO₄, can be prepared in a simple way by addition of phosphoric acid to the corresponding oxide or hydroxide, for example magnesium oxide or hydroxide, which yields phosphate salt. The phosphate salt obtained can subsequently be added (optionally after filtering and washing).

The quantity of phosphate salt to be used preferably lies between 0.5 and 3 phosphate salt equivalents, in particular between 0.8 and 1.2 equivalents, related to the quantity of ammonia that is formed during the reaction.

Examples of suitable enzymes that can be used are the enzymes that are usually used in hydantoinase-carbamoylase processes, for example enzymes derived from the genus Pseudomonas, in particular Pseudomonas fluorescens, putida or desmolytica, Achromobacter, Corynebacterium, Bacillus, in particular Bacillus brevis or Bacillus stearothermophilus, Brevibacterium, Microbacterium, Artrobacter, Agrobacterium, in particular Agrobacterium tumefaciens or radiobacter, Acrobacter, Klebsiella, Sarcina, Protaminobacter, Streptomyces, Actinomyces, Candida, Rhodotorula, Pichia or Paecilomyces.

The enzymatic reaction can be carried out at a pH that lies between pH 5 and pH 9 and is preferably carried out at a pH that lies between pH 6 and 8. The temperature at which the enzymatic reaction is carried out preferably lies between 0 and 50°C, in particular between 20 and 40°C. Upon completion of the enzymatic reaction the reaction mixture can be recovered in various ways.

A suitable recovery for example takes place by acidifying the reaction mixture to a pH between 0 and 3, preferably between 0.5 and 1.5, followed by removal of the biomass. After increasing the pH to for example a value between 3 and 5, preferably between 3.5 and 4.5, the D-amino acid can be separated, for example by filtration or centrifugation. After increasing the pH to a value between 5 and 11, preferably between 7 and 10, the corresponding ammonium phosphate salt formed from the phosphate salt can be separated for example via centrifugation or filtration.

Another suitable recovery takes place for example by increasing the pH to a value between 9 and 11, preferably between 9.5 and 10.5, after which the corresponding solid ammonium phosphate salt formed from phosphate salt can be filtered off. From the resulting mother liquor the biomass is subsequently removed, for example by means of microfiltration or ultrafiltration. After acidifying to a pH of for example between 3 and 6, preferably between 4.5 and 5.5, solid D- amino acid can subsequently be isolated, for example by means of filtration. The resulting ammonium phosphate salt can subsequently simply be converted in a known way into the phosphate salt by dry heating of the ammonium phosphate salt, with ammonia being liberated. Another method is to heat a slurry of the ammonium phosphate salt at a pH > 8.5, in particular between 9 and 11 , with ammonia being liberated. Yet another method is to wash the magnesium ammonium phosphate salt with a mineral acid, for instance sulphuric acid, keeping the pH between 4.5 and 6.5, preferably between 5.5 and 6. Accordingly the salt of ammonium and the mineral acid is obtained and Mg hydrophosphate can be recovered.

In US-A-4,460,555 and US-A-4,650,857 from 1984 and 1985 specific magnesium hydrophosphate particles are described as ammonia scavengers. These patent publications are particularly concerned with such particles for use in enzymatic dialysis systems for the removal of urea with the aid of urease by scavenging ammonia liberated from the urea. The total quantity of ammonia that is liberated here is, however, relatively low. The use of such particles in hydantoinase/carbamoylase processes for the prevention of enzyme inhibition has, however, never been suggested in the time elapsed since then. The invention is particularly suitable for use in the preparation of enantiomerically enriched amino acids via the so-called hydantoin route, which involves the preparation of N-carbamoylamino acid enriched in the D-enantiomer from the corresponding hydantoin with the aid of a hydantoinase, optionally in combination with a racemase, followed by the decarbamoylation with the aid of D- carbamoylase, wherein the decarbamoylation is the overall reaction rate determining step. It has been found that the presence of phosphate salts and/or ammonium phosphate salts has no inhibitive or denaturing effect on hydantoinase, carbamoylase and/or racemase. In the known processes the pH at which these conversions take place mostly lies between 7 and 8, since at higher pH values virtually complete enzyme inhibition occurs due to the presence of NH₃. A disadvantage of carrying out the reactions at an approximately neutral pH is that in the preparation of almost all amino acids, in particular of aliphatic amino acids, slow racemisation of the hydantoin enantiomer remaining behind takes place.

It has now been found that when the reactions according to the invention are carried out at higher pH, for example at a pH between 7.0 and 9.0, preferably between 7.5 and 8.5, almost no enzyme inhibition occurs while racemisation of the unwanted enantiomer does occur. As a result a yield can be achieved that is much higher than the 50% that is theoretically the maximum possible without racemisation. The process according to the invention can also be used in resolution processes in which a DL-N-carbamoylamino acid is converted to the corresponding amino acid enriched in the D-enantiomer and the non- converted L- N-carbamoylamino acid enriched in the enantiomer with the aid of a microorganism that contains a D-selective hydantoin-hydrolysing and a N-carbamoyl- amino acid-hydrolysing enzyme. As the reaction can be carried out at an elevated pH (for example between 7.5 and 9), there is less loss of L-N carbamoylamino acid, for at elevated pH the hydantoinase reaction does not proceed.

Examples:

Example I

34 g DL-p-hydroxyphenylglycine hydantoin and 34 g MgHPO₄-3H₂O was added to 200 ml water. After the pH of the reaction mixture had been adjusted to pH = 7.2 with the aid of 5 N NaOH the reaction was started by addition of 34 ml Agrobacterium radiobacter cell suspension. The reaction was carried out under a nitrogen atmosphere at 40°C. The pH of the reaction mixture was held constant at pH 7.2 by addition of 5 N NaOH. By means of HPLC analysis it was established that a conversion of >99% was reached already after 10 hours hydrolysis. Comparative experiment 1

Under comparable conditions as described in example 1 34 g DL-p-hydroxyphenylglycine hydantoin was hydrolysed, however, in the absence of the phosphate salt. The reaction was started by addition of 34 ml of the same Agrobacterium radiobacter cell suspension. The reaction was carried out under a nitrogen atmosphere at 40°C. The pH of the reaction mixture was kept constant at pH = 7.2 by means of an automatic titration with 1.3 M H₃PO₄. At regular times the progress of the reaction was checked by means of an HPLC analysis. It was recorded that after approx. 10 hours hydrolysis a conversion of approx. 57% was obtained. The conversion measured after 30 hours amounted to >99%.

Example II

A hydrolysis of DL-p-hydroxyphenylglycine hydantoin was carried out by adding 122 g of this compound with 122 g MgHPO₄-3H₂O to 575 ml water. The pH of this mixture was adjusted to pH = 7.2 with 5 N NaOH after which nitrogen gas was passed through for 1 hour. The enzymatic conversion was started by addition of 8 ml Agrobacterium radiobacter cell suspension. The pH of the reaction mixture was kept constant at pH = 7.2 by addition of NaOH (25 wt.%). In total an NaOH consumption of 19 g was recorded. After 95 hours hydrolysis a conversion of 99.3% was measured.

Comparative experiment 2

Under comparable conditions as described in example II p- hydroxyphenylglycine hydantoin (122 g) was added to 575 ml water at 40°C. After the pH was adjusted to 7.2 with 5 N NaOH, nitrogen gas was passed through for 1 hour. The reaction was started by addition of 30 ml of the same Agrobacterium radiobacter cell suspension. The pH was held constant at pH 7.2 with a 33 wt.% H₃PO₄ solution. After 95 hours hydrolysis a conversion of 98.7% was measured. The conversion measured after 97.5 hours hydrolysis amounted to 99.3%.

Example III

At 40°C 176 gram DL-p-hydroxyphenylglycine hydantoin and 176 g MgHPO₄-3H₂O was added to 510 ml water. This reaction mixture was inertised for 1 hour with the aid of nitrogen. The enzymatic conversion was started by addition of 15 ml of an Agrobacterium radiobacter cell suspension. The pH of the reaction mixture was kept constant at pH = 7.2 by means of an automatic titration with 5 N NaOH. By means of HPLC analysis it could be established that the conversion after 120 hours hydrolysis amounted to 99.8%.

Comparative experiment 3

At 40°C 176 gram DL-p-hydroxyphenylglycine hydantoin was added to 557 mL water and the pH was adjusted to 7.2 with 5 N NaOH. This reaction mixture was inertised for 1 hour with the aid of nitrogen. Then 56 ml of the same Agrobacterium radiobacter cell suspension was added. The pH of the reaction mixture was kept constant at pH = 7.2 by means of an automatic titration with 33 wt.% H₃PO₄. By means of HPLC analysis it could be established that the conversion after 146.5 hours hydrolysis was 96.5% and after 180 hours 99.8%.

Example IV A reaction mixture obtained as described in example II was acidified with around 130 g H₂SO₄ (98 wt.%) to pH = 1 after which the cell residues were removed by means of a microfiltration. The retentate was diafiltered with 100 g water. The collected aqueous phases were partially evaporated to a volume of approximately 600 ml. About 63 g NaOH (50 wt.%) was added to the resulting residue until the pH was 3.5, and the D-p-hydroxyphenylglycine formed was separated and washed a few times. To the filtrate of this step approximately 53 g NaOH (50 wt.%) was added to a pH of 8.5 after which the MgNH₄PO₄ formed could be separated.

The MgNH₄PO₄ was washed twice with 50 ml water after which it was suspended in water. Next, a mixture of water and ammonia was evaporated under reduced pressure.

Ultimately 101.7 g D-p-hydroxyphenylglycine and 118 gram MgHPO₄-3H₃O was obtained.

Example V

The pH of a reaction mixture obtained as described in example II was adjusted to pH = 10 with approximately 70 g NaOH (50 wt.%). The reaction mixture was filtered after which the residue was washed twice with 100 ml water. The filtrate of this step was led through a microfiltration set-up to remove any cell residues. The retentate was washed a few times. The permeate was then acidified with around 40 g H₂SO₄to a pH of 3.5, upon which D-p-hydroxyphenylglycine crystallises. After separating, washing and drying a yield of 102 g D-p- hydroxyphenylglycine was obtained.

Example VI

The conversion of DL-p-hydroxyphenylglycine hydantoin to D-(-)- p-hydroxyphenylglycine was carried out at various pH values.

34 g (177 mmol) DL-p-hydroxyphenylglycine hydantoin and 34 g MgHPO₄-3H₂O was added to 200 ml water. The reaction mixture was brought to the desired pH with the aid of 5 N NaOH after which the reaction was started by addition of 3.0 ml of an Agrobacterium radiobacter cell suspension. The reaction was carried out under a nitrogen atmosphere at 40°C. The pH of the reaction mixture was kept at the desired pH by means of an automatic titration with 5 N NaOH. The results of these experiments are presented in table 1. This table gives the reaction time at which a conversion of > 99% was achieved.

Table 1

Example VII

At 40°C 30 g DL-N-carbamoyl-p-hydroxyphenylglycine and 34 g MgHPO₄-3H₂O was added to 200 ml water. The pH of the reaction mixture was adjusted to pH 8.0 with about 15 g 42% KOH. Next, under a nitrogen atmosphere, 13 ml of an Agrobacterium radiobacter cell suspension was added. The pH of the reaction mixture was kept constant at pH 8.0. The composition of the reaction mixture was monitored by means of HPLC analysis. After a reaction time of 27 hours a D-p-hydroxyphenylglycine concentration of 4.3 wt.% was measured and an L-N-carbamoyl-p-hydroxyphenylglycine concentration of 5.4 wt. %. The composition of the reaction mixture subsequently remained constant.

Example VIII 15 g DL-valine hydantoin and 20 g MgHPO₄-3H₂O was added to

200 ml water. After the pH was adjusted to 8 by addition of 5 N NaOH the reaction was started by addition of 15 ml Agrobacterium radiobacter cell suspension. The reaction was carried out at 40°C under a nitrogen atmosphere. The pH of the reaction was kept constant at pH = 8 by addition of 5 N NaOH. After 16 hours hydrolysis a D-valine concentration of 52 g/l was measured, which corresponds to a conversion of > 99% on the basis of the DL-valine hydantoin.

Claims

C L A I M S

1. Process for the preparation of a chiral amino acid enriched in the D- enantiomer, in which a mixture of the enantiomers of the corresponding

N-carbamoylamino acid is brought into contact with a D-carbamoylase with ammonia being liberated, characterised in that the ammonia is removed with the aid of a bivalent metal salt of a phosphate ion, a monohydrogen phosphate ion or a dihydrogen phosphate ion.

2. Process according to claim 1, wherein the enzymatic decarbamoylation is carried out in the presence of a bivalent metal salt of a phosphate ion, a monohydrogen phosphate ion or a dihydrogen phosphate ion.

3. Process according to claim 1, wherein the reaction mixture is contacted with the bivalent metal salt of a phosphate ion, monohydrogen phosphate ion or dihydrogen phosphate ion, via an external loop, after separation of the solid present.

4. Process for the preparation of a chiral amino acid enriched in the D- enantiomer in which the corresponding hydantoin is enzymatically converted with the aid of a hydantoinase into the corresponding N- carbamoylamino acid, which is subsequently converted into the amino acid enriched in the D-enantiomer using the method according to one of claims 1-3.

5. Process according to claim 4 in which both steps are carried out in a vessel in the presence of both a hydantoinase and a D-carbamoylase.

6. Process according to claim 4 or 5 in which the pH of the reaction mixture lies between 7.0 and 9.0.

7. Process according to any one of claims 1-6, in which magnesium monohydrogen phosphate is used as the bivalent metal salt of a phosphate ion.

8. Process according to any one of claims 1-7, in which the reaction mixture obtained is subsequently subjected to a pH increase to a pH between 9.5 and 10.5, separation of solid ammonium phosphate salt formed, pH reduction of the mother liquor to a pH between 3.5 and 4.5 and separation of the solid D-amino acid.

9. Process according to any one of claims 1-7, in which the reaction mixture obtained is subsequently subjected to a pH reduction to a pH between 0.5 and 1.5, separation of the biomass, a further pH increase to a pH between 3.5 and 4.5 and separation of the solid D-amino acid.

10. Process according to claim 9 in which the mother liquor obtained is subsequently subjected to a pH increase to a value between 7 and 10 and the solid ammonium phosphate salt formed is separated.

11. Process for the recovery of magnesium monohydrogen phosphate wherein ammonium phosphate is contacted with a mineral acid at a pH between 4.5 and 6.5 and the magnesium monohydrogen phosphate is separated from the salt of ammonia and the mineral acid.

12. Process according to claim 11 wherein the pH is kept between 5.5 and 6.

13. Process according to claim 11 or 12 wherein the mineral acid is sulphuric acid.