NL1017250C1 - Process for the preparation of enantiomerically enriched amino acids. - Google Patents

Description

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Process for the preparation of enantiomerically enriched 5-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 contacted with a D-carbamoylase in which ammonia is released.

D-amino acids are important building blocks for biologically active preparations such as l-lactam antibiotics, peptide hormones and pesticides. A widely used method of preparation for these "unnatural" amino acids is a method in which the corresponding DL-5-substituted hydantoin is enantioseively hydrolysed by a hydantoinase to the corresponding N-carbamoylamino acid. This N-carbamoylamino acid can be enzymatically converted to the corresponding D-amino acid.

A drawback of the known method is that, in order to realize a certain reaction time, relatively large amounts of biocatalyst are required because it is generally known that the enzyme 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. Described is (Olivieri et. Al. (1981) Biotechnology and 25 Bioengineering, vol. 23, 2173-2183; Runser (1990) Applied Microbiology and Biotechnology, 33, 382-388; 1 Π) i \ - 2 -

Louwrier (1996) Enzyme and Microbial Technology, 19, 562-571) that the concentration at which inhibition by ammonia occurs is very low (~ 10 mM). For the known processes this means that at a concentration of 10 mM ammonia the carbamoylase is active at half the maximum speed.

At a concentration of 20 mM ammonia, the speed of the carbamoylase is only 1/3 of the maximum speed. It will be clear that under industrially relevant conditions where high product concentrations, for example> 500 mM, are desirable, the speed of the carbamoylase will be very strongly inhibited.

Lower product concentrations where the activity of the carbamoylase is practically maximal are economically unattractive due to the low production capacity.

Various methods have been described in the literature to keep the ammonia concentration low during the enzymatic conversion. An example of in-situ removal of ammonia is described by Kim & Kim, 15 (1995) Enzyme and Microbial Technology, 17, 63-67 in which an adsorbent is used during the preparation of Dp-hydroxyphenylglycine using hydantoinase and carbamoylase (AD300NS, a silicate complex from Tomita Pharmaceutical Co. (Japan)) for the removal of ammonia. A disadvantage of this method is that the adsorbent is expensive and must therefore be recycled.

According to another method described in J6 1285-996-A (1986), the ammonia formed is removed by distillation. However, this ammonia removal method is only effective at high pH of the reaction mixture. However, the enzyme is not active at such a high pH.

When ammonia is removed by distillation at neutral pH, between pH 7-7.5, even if more than 90% of the reaction volume is removed, 60 to 90% of the ammonia formed remains in the reaction mixture. Efficient removal at neutral pH is therefore not possible.

The invention now provides a simple and economically attractive process in which less biocatalyst has to be used or a shorter reaction time is realized.

This is achieved according to the invention by removing the ammonia with the aid of a divalent metal salt of a phosphate, a monohydrogen phosphate or a dihydrogen phosphate ion; in the further description 35 simply referred to as phosphate salt. To this end, for example, the enzymatic 3 reaction can be carried out in the presence of a phosphate salt. Moreover, it has surprisingly been found that the reaction mixture remains well stirrable even at high slurry concentrations.

Another embodiment is formed, for example, by passing the reaction mixture through, for example, a loop after separation of the undissolved reaction components, through, for example, a second reactor, or a column or filter containing the phosphate salt. The ammonia present in the reaction mixture is then bound to the phosphate salt, the corresponding ammonium phosphate salt being formed, after which the remaining liquid, which for example still contains enzyme, is returned to the enzymatic decarbamoylation reaction.

It has been found that no or much less enzyme inhibition occurs. This is all the more surprising because it is known that divalent metals can disturb carbamoylase catalyzed reactions at low concentrations (1 to 10 μ molar).

Suitable divalent metal ions are, for example, magnesium, cobalt, calcium, manganese, zirconium or ruthenium ions. For economic reasons, magnesium monohydrogen phosphate (MgHPO 4.), Also called magnesium hydrophosphate, is preferably used. MgHPO 4 fits pH with the optimum process conditions and is easy to prepare from inexpensive raw materials. The phosphate salt can also be formed in situ.

The phosphate salt, for example MgHPO 4, can be prepared in a simple manner by adding phosphoric acid to the corresponding oxide or hydroxide, for example magnesium oxide or hydroxide, whereby phosphate salt is formed. The resulting phosphate salt can then be added (optionally after filtering and washing).

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

Suitable enzymes that can be used are, for example, the enzymes commonly used in hydantoinase-carbamoylase processes, for example enzymes from the genus Pseudomonas, in particular Pseudomonas fluorescens, putida or desmolytica, Achromobacter, Corynebacterium, Bacillus, in particular Bacillus brevis or - 4 -

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 between pH 5 and pH 9 and is preferably carried out at a pH between pH 6 and 8. The temperature at which the enzymatic reaction is carried out is preferably between 0 and 50. ° C, in particular between 20 and 40 ° C.

After the enzymatic reaction has ended, the reaction mixture can be worked up in various ways.

A suitable work-up is, for example, via acidification of the reaction mixture to a pH between 0 and 3, preferably between 0.5 and 1.5, followed by removal of the biomass. After raising 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 off, for example by filtration or centrifugation. After raising 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, for example, be separated via centrifugation or filtration.

Another suitable work-up proceeds, for example, via a pH increase 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. The biomass is then removed from the mother liquor obtained, for example with the aid of micro- or ultrafiltration. After acidification to a pH, for example, between 3 and 6, preferably between 4.5 and 5.5, solid D-amino acid can then be isolated, for example by filtration.

The resulting ammonium phosphate salt can then easily be converted in known manner into the phosphate salt by dry heating of the ammonium phosphate salt, thereby evading ammonia. Another method is to heat a slurry of the ammonium phosphate salt at a pH> 8.5, in particular between 9 and 11, thereby evading ammonia.

US-A-4460555 and US-A-4650857 from 1984 and 1985 describe specific magnesium hydrophosphate particles as ammonia scavengers. These patent publications are particularly directed to such particles for use in enzymatic dialysis systems for the removal of urea with the aid of urease by means of capturing ammonia released from the urea. However, the total amount of ammonia that is released here is relatively low. However, the use of such particles in hydantoinase / carbamoylase processes to prevent enzyme inhibition has never been suggested in the time since.

The invention can be used particularly well in the preparation of enantiomerically enriched amino acids via the so-called hydantoin route wherein N-carbamoyl-amino acid enriched in the D-enantiomer enriched with the corresponding hydantoin is prepared with the aid of a hydantoinase, optionally in combination with a racemase followed by decarbamoylation using D-carbamoylase. It has been found that the presence of phosphate salts and / or ammonium phosphate salts has no inhibitory or denaturing effect on hydantoinase, carbamoylase and / or racemase. In the known processes, the pH at which these conversions take place is usually between 7 and 8, since at higher pHs enzyme inhibition occurs due to the presence of NH 3. A disadvantage of carrying out the reactions at approximately neutral pH is that in the preparation of almost all amino acids, in particular of aliphatic amino acids, there is no or insufficient racemization of the remaining hydantoin enantiomer.

It has now been found that when the reactions according to the invention are carried out at a higher pH, for example at a pH between 7.0 and 9.0, preferably between 7.5 and 8.5, virtually no enzyme inhibition occurs while there is racemization. of the undesired enantiomer. Consequently, a yield can be realized that is much higher than the 50% that is theoretically possible without racemization.

The method according to the invention can also be used in resolution processes in which a DL-N-carbamoylamino acid is converted with the aid of a microorganism containing a D-selective hydantoin hydrolyzing and N-carbamoylamino acid hydrolyzing enzyme to the corresponding amino acid enriched in the D-enantiomer and the unreacted LN-carbamoylamino acid enriched in the enantiomer. Because the reaction can be carried out at an elevated pH (for example between 7.5 and 9), less loss of L-N-carbamoyl amino acid occurs, since at a higher pH the hydantoinase reaction does not proceed.

35 "I".

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Examples Example I

To 200 ml of water was added 34 g of DL-p-hydroxyphenylglycine hydantoin and 34 g of MgHPO 4 -3H 2 O. After the pH of the reaction mixture was adjusted to pH = 7.2 using 5 N NaOH, the reaction was started by adding 34 ml of 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 the addition of 5 N NaOH. It was determined by HPLC analysis that a conversion of> 99% was achieved after 10 hours of hydrolysis.

Comparative Experiment 1 Under similar conditions as described in Example 1, 34 g of DL-p-hydroxyphenyl glycine hydantoin was hydrolyzed, however, in the absence of the phosphate salt. The reaction was started by adding 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 H3 PO4. The progress of the reaction was checked at regular intervals by an HPLC analysis. It was observed that after approximately 10 hours of hydrolysis a conversion of approximately 57% was obtained. The conversion measured after 30 hours was> 99%.

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EXAMPLE II

A hydrolysis of DL-p-hydroxyphenyl glycine hydantoin was carried out by adding 122 g of this compound with 122 g of MgHPO 4 -3H 2 O to 575 ml of 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 adding 8 ml of Agrobacterium radiobacter cell suspension. The pH of the reaction mixture was kept constant at pH = 7.2 by the addition of NaOH (25% by weight). In total, a NaOH consumption of 19 g was recorded. After 95 hours of hydrolysis a conversion of 99.3% was measured.

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Comparative experiment 2

Under similar conditions as described in Example II, p-hydroxyphenyl glycine hydantoin (122 g) was added to 575 ml of 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 adding 30 ml of the same Agrobacterium radiobacter cell suspension. The pH was kept constant at pH 7.2 with a 33 wt. % H 3 PO 4 solution. After 95 hours of hydrolysis, a conversion of 98.7% was measured. The conversion, measured after 97.5 hours of hydrolysis, was 99.3%.

EXAMPLE III

To 510 ml of water was added 40 g of DL-p-hydroxyphenylglycine hydantoin and 176 g of MgHPO 4 -3H 2 O at 40 ° C. This reaction mixture was inertized with nitrogen for 1 hour. The enzymatic conversion was started by adding 15 ml of an Agrobacterium radiobacter cell suspension. The pH of the reaction mixture was kept constant at pH 7.2 by automatic titration with 5 N NaOH. It could be determined by HPLC analysis that the conversion after 120 hours of hydrolysis was 99.8%.

Comparative experiment 3

To 557 mL of water was added 176 grams of DL-p-hydroxyphenylglycine hydantoin at 40 ° C and adjusted to pH 7.2 with 5 N N NaOH. This reaction mixture was inertized with nitrogen for 1 hour. After this, 56 ml of the same Agrobacterium radiobacter cell suspension was added. The pH of the reaction mixture was adjusted by means of an automatic titration with 33 wt. % H 3 PO 4 kept constant at pH = 7.2. By means of HPLC analysis it could be established that the conversion was 96.5% after 146.5 hours and 99.8% after hydrolysis for 180 hours.

EXAMPLE IV

A reaction mixture obtained as described in Example II was acidified with approximately 130 g of H 2 SO 4 (98% by weight) to pH = 1, after which the cell debris was removed by means of a microfiltration. The retentate was diafiltered with 100 g of water. The collected water phases were partially evaporated to a volume of approximately 600 ml. Approximately 63 g of NaOH (50 wt.%) Were added to the resulting residue to a pH of 3.5 and the D-p-hydroxyphenylglycine formed was separated and washed several times. About 53 g of NaOH (50% by weight) was added to the filtrate of this step to a pH of 8.5, after which the MgNH 4 PO 4 formed could be separated.

The MgNH 4 PO 4 was washed twice with 50 ml of water after which it was suspended in water. A mixture of water and ammonia was then evaporated under reduced pressure.

1 o Finally, 101.7 g of D-p-hydroxyphenylglycine was obtained and 118 g of MgHPO 4 -3H30.

Example V

A reaction mixture obtained as described in Example II was adjusted to pH = 10 with approximately 70 g of NaOH (50% by weight). The reaction mixture was filtered and the residue was washed twice with 100 ml of water. The filtrate from this step was passed through a microfiltration arrangement to remove any cell debris present. The retentate was washed several times. The permeate was then acidified with about 40 g of H 2 SO 4 to a pH of 3.5 at which D-p-hydroxyphenyl glycine crystallizes. After separation, washing and drying, a yield of 102 g of D-p-hydroxyphenyl glycine was obtained.

Example VI

The conversion of DL-p-hydroxyphenyl glycine hydantoin to D - (-) - p-hydroxyphenyl glycine was performed at various pH values.

To 200 ml of water was added 34 g (177 mmol) of DL-p-hydroxyphenylglycine hydantoin and 34 g of MgHPO 4 -3H 2 O. The reaction mixture was adjusted to the desired pH using 5 N NaOH after which the reaction was started by adding 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 maintained at the desired pH by means of an automatic titration with 5 N NaOH. The 3 results of these experiments are shown in Table 1. This table shows the reaction time in which a conversion of> 99% was achieved.

Table 1 pH reaction time (hour) for> 99% conversion __ _ __ _ __ _ 7/7 - 7β 88 5

EXAMPLE VII

30 g of DL-N-carbamoyl-p-hydroxyphenylglycine and 34 g of MgHPO 4 -3H 2 O were added to 200 ml of water at 40 ° C. The pH of the reaction mixture was adjusted to pH 8.0 with approximately 15 g of 42% KOH. Subsequently, 13 ml of an Agrobacterium radiobacter cell suspension was added under a nitrogen atmosphere. The pH of the reaction mixture was kept constant at pH 8.0. The composition of the reaction mixture was monitored by HPLC analysis. After a reaction time of 27 hours, a D-p-hydroxyphenylglycine concentration of 4.3 wt. % and an L-N-carbamoyl-15 p-hydroxyphenylglycine concentration of 5.4 wt. %. The composition of the reaction mixture then remained constant.

EXAMPLE VIII

15 g of DL-valine hydantoin and 20 g of MgHPO 4 -3H 2 O were added to 200 ml of water. After the pH was adjusted to 8 by adding 5 N NaOH, the reaction was started by adding 15 ml of 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 the addition of 5 N NaOH.

After 16 hours of hydrolysis, a D-valine concentration of 52 g / l was measured, which corresponds to a conversion of> 99% based on the DL-valine hydantoin.

Claims (10)

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 whereby ammonia is released, characterized in that the ammonia is removed with the aid of a divalent metal salt of a phosphate, a monohydrogen phosphate or a dihydrogen phosphate ion. The method of claim 1, wherein the enzymatic decarbamoylation is carried out in the presence of a divalent metal salt of a phosphate, a monohydrogen phosphate or a dihydrogen phosphate ion. The method of claim 1, wherein the reaction mixture is contacted via an external loop, after separation of the solid present, with the divalent metal salt of a phosphate, monohydrogen phosphate or dihydrogen phosphate ion. 4. Process for the preparation of a chiral amino acid enriched in the D-enantiomer, wherein the corresponding hydantoin is enzymatically converted with the aid of a hydantoinase into the corresponding N-carbamoyl-amino acid which is subsequently processed by the method according to any one of claims 1- 3 is converted to the amino acid enriched in the D-enantiomer. 2 5 The method of claim 4 wherein both steps are performed in a pot in the presence of both a hydantoinase and a D-carbamoylase. The method according to claim 4 or 5, wherein the pH of the reaction mixture is between 7.0 and 9.0. Process according to any one of claims 1-6, wherein magnesium monohydrogen phosphate is used as the divalent metal salt of a phosphate ion. 8. A method according to any one of claims 1-7, wherein the resulting reaction mixture is subsequently subjected to a pH increase to a pH between 9.5 and 10.5, separation of formed solids if any. Ammonium phosphate salt, pH reduction of the mother liquor to a pH between 3.5 and 4.5 and separation of the solid D-amino acid. 9. A method according to any one of claims 1-7, wherein the resulting reaction mixture is subsequently subjected to a pH reduction to a pH between 0.5 and 1.5, separation of the biomass, further pH increase to a pH between 3 5 and 4.5 and separation of the solid D-amino acid. The method according to claim 9, wherein the resulting mother liquor is then subjected to a pH increase to a value between 7 and 10 and the solid ammonium phosphate salt formed is separated.