CZ20032077A3 - Process for preparing enantiomer-enriched amino acids - Google Patents

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

The present invention relates to a process for preparing an amino acid enriched in the D-enantiomer, wherein a mixture of the enantiomers of the corresponding N-carbamoylamino acids is contacted with D-carbaraoylase and ammonia is released.

BACKGROUND OF THE INVENTION. ,

D-amino acids are important building blocks for biologically active agents such as β-lactam antibiotics, peptide hormones and pesticides.

In the prior art, the preparation of these non-natural amino acids generally involves a process in which the corresponding DL-5-substituted hydantoin is enantiated selectively hydrolyzed with hydantoinase to give the corresponding N-carbamoylamino acid. This N-carbamoylamino acid is converted to the corresponding D'-amino acid by enzymatic cleavage.

A disadvantage of this known prior art process is that if the aim of the solution is to carry out the process in a certain reaction interval, then a relatively large amount of biocatalyst is required, as it is well known that the enzyme responsible for hydrolysis carbamoylamino acid (carbamoylase, also referred to as N-carbamoyl-D-amino acid amide hydrolase) is strongly inhibited by the reaction product, ammonia. The concentration at which inhibition due to ammonia occurs is reported in the prior art

p. i · '· <»» »» »» »» »» »

For techniques, see 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, and this concentration is known to be very low (approximately 10 mM). When these known processes are carried out, this means that at an ammonia concentration of 10 mM, the carbamoylase is only effective at half its maximum value. At an ammonia concentration of 20 mM, the efficacy of this carbamoylase is. , * ⁷ ; . - 1 only 1/3 of its maximum value. From above; It will be appreciated that under the conditions of industrial practice, when a high product concentration, for example a concentration of &gt; 500 mM, is required, a very rapid inhibition of carbamoylase occurs. The lower concentration of the product (in the 10 mM range) at which the carbamoylase activity is almost maximal is not economically attractive because of it. low production capacity J occurs. . . ·

Various prior art methods for maintaining concentration are described in the prior art literature:

ammonia at the lowest level # during the enzymatic conversion. Examples of such tn situ removal of ammonia performed · je'možno, noted by Kim & Kim (1995), Enzyme and Microbial Technology, 17, 63-67, wherein: · when this procedure is used; kfodstraňování ammonia during ^-> the process the preparation of Dβ-hydroxyphenylglycine with hydantoinase and carbamoylase adsorbent (AD300NS, a silicate complex of Tornay Pharmaceuticals Co.).

(Japan). The disadvantage of this process is that this adsorbent is expensive and therefore needs to be recycled.

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A further procedure is described in J 61.285.996-A (1986), wherein the ammonia formed is removed by distillation. However, this ammonia removal process is effective only when the reaction mixture has a high pH. At this high pH, however, the carbamoylase enzyme is inactive. When ammonia is removed by distillation at neutral pH, that is. at a pH in the range of 7 'to 7.5, then 60 to 90% of the ammonia thus formed remains in the reaction mixture even if more than 90% of the reaction volume is removed. From above. It is clear that effective removal at neutral pH is not possible. Λ ‘.

SUMMARY OF THE INVENTION. ₄ '- ♦'

The present invention has now found a simple and economically attractive process requiring less biocatalyst or shorter reaction time. * \

These results can be achieved by removing the ammonia with the aid of a divalent metal salt and a phosphate salt. or a dihydrogen phosphate ion or dihydrogen phosphate ion, hereinafter referred to as the phosphate ion for ease of reference. Thus, it can simply be said that this enzymatic reaction can be carried out (for example) in the presence of a phosphate salt. In addition, it has been unexpectedly found that the reaction mixture remains readily stirrable even at high suspension concentrations. ·. .

Other embodiments of the process of the present invention include:

in that the reaction mixture is, for example, passed through a circuit after separation of the undissolved reactants, for example, into a second reactor or column or filter in which the phosphate salt is present. The ammonia contained in the reaction mixture then binds to the phosphate salt to form the corresponding ammonium phosphate salt, whereupon the remaining liquid, in which the enzyme is still contained, is returned to the reaction vessel for enzymatic decarbamoylation. ’

According to the present invention ^4, it was found that during this procedure there will be no enzyme inhibition or to inhibit this will only occur to a much lesser extent than is the case in the procedures of the prior art.

This is all the more surprising since it is known that divalent metals can influence carbamoylase-catalyzed reactions even at low concentrations (ranging from 1 to 10 μ moles). in

Examples of suitable divalent metal ions include magnesium, cobalt, calcium, manganese, zirconium or ruthenium ions. For economic reasons, magnesium hydrogen phosphate MgHPO4, which is also referred to as the middle phc ', is preferably used. or magnesium phosphate secondary · MgSO4 magnesium phosphate is suitable from the viewpoint of ««. · Adherence to optimum operating conditions regarding the resulting pH value and ease of preparation from cheap raw materials. The phosphate salt may also be prepared in situ.

-1%.

The phosphate salt, such as MgHPO 4, can be prepared in a simple manner by adding phosphoric acid to the corresponding oxide or hydroxide, for example magnesium oxide or magnesium hydroxide, to give the desired phosphate salt. The phosphate salt can then be added immediately to the process (optionally after filtering and washing).

The amount of the phosphate salt to be used in the process ranges from 0.5 to 3 equivalents of the phosphate salt, based on the amount of ammonia formed during the reaction, preferably from 0.8 to 1.2 equivalents .

Examples of suitable enzymes which can be used in the process include enzymes which can be used

I usually use hydantoinase-carbamoylase processes, such as enzymes deprotected from, to carry out processes. j strains of Pseudomonas, in particular Pseudomonas fluorescens, putida or desmolytica, Achromobacter, Corynebacterium, Bacillus, in particular Bacillus brevis or Bacillus stearothemophilus, Brevibacterium, Microbacterium, Artrobacter, Agrobacterium, in particular Agrobacterium radiefacterens,

Acrobacter, Klebsilla, Sarcina, Protaminobacter,

Streptomyces, Actinomyces, Candida, Rhodotorula, Pichia, or Paecilomyces.

The enzymatic reaction may be carried out at a pH ranging from pH 5 to pH 9, preferably at a pH ranging from 6 to 8. The temperature at which the enzymatic reaction is carried out is preferably in the range from 0 to 50 ° C, in particular in the range from 20 to 40 ° C.

Upon completion of this enzymatic reaction, the enzyme mixture obtained is regenerated in various ways.

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For example, one suitable regeneration method is to acidify the reaction mixture to a pH of from 0 to 3, preferably to a pH of from 0.5 to 1.5, followed by removal of the biomass. By increasing the pH, for example, to a value in the range of from 3 to 5, preferably to a value in the range of from 3.5 to 4.5, the D-amino acid can be removed, for example by filtration or centrifugation. After increasing the pH. to a value in the range of from 5 to 11, preferably to;

a value in the range of from 7 to 10 produces a corresponding ammonium phosphate salt from the phosphate salt, which can be removed, for example, by centrifugation or filtration. j

Another regeneration method is carried out, for example, by raising the pH to a value in the range of from 9 to 11, preferably (in the range of from 9.5 to 10.5, whereupon the solid thus formed the ammonium phosphate salt formed from the corresponding phosphate salt is filtered off.

Biomass is subsequently removed from the resulting mother liquor thus obtained. for example by microfiltration or ultrafiltration. After acidification to a pH of, for example, from 3 to 6, preferably from 4.5 to 5.5, the solid is separated

The D-amino acid is separated in the next stage, for example, by filtration.

The resulting ammonium phosphate salt thus obtained can be converted into the phosphate salt in a conventional manner in a simple manner by dry heating of the ammonium phosphate salt in a subsequent manner, releasing ammonia. Another method consists in heat treating a suspension of the ammonium phosphate salt at a pH > 8.5, in particular 9 99 9. 7 '' <· · · 9 9 9; 9.-9 9, / - · * · ··. ·· · · ·. 9 9999 99 99 in the range from 9 to 11, releasing ammonia again.

Yet another method involves washing a salt, ammonium magnesium phosphate, with a mineral acid, for example an acid

Sulfuric acid, maintaining a pH in the range of 4.5 to 6.5, preferably in the range of 5.5 to 6. The process results in an ammonium salt of the mineral acid and magnesium hydrogen phosphate.

U.S. Pat.

US-A-4,460,555 and US-A-4,650,857 of 1984 and 1985 disclose specific magnesium hydrogen phosphate particles as ammonia scavenging particles. These patents, however, are particularly directed to the use of these particles in enzymatic dialysis systems in which they are used to remove urea by means of ^3- urease by trapping ammonia released from urea. However, the total amount of ammonia released in this manner is relatively small.

The process of the present invention is particularly suitable for use in the preparation of enantiomerically enriched amino acids via the so-called hydantoin pathway, which comprises preparing the N-carbamino amino acid enriched for the D-enantiomer from the corresponding hydantoin using hydantoinase, optionally in combination with racemase decarbamoylation with D-carbamoylase, the decarbamoylation being a step affecting the overall reaction rate. 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

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4 4 4 4 4 4 4 «44 • 4 4, 44 44 4« 44> * · »· and • 9 racemase. In the known prior art, the pH of these conversions is usually in the range of 7 to 8, because at higher pH values the enzyme is almost completely inhibited due to the presence of ammonia. The disadvantage of carrying out these reactions at approximately neutral pH is that in the preparation of almost all amino acids, especially aliphatic amino acids, there is a slow racemization of the hydantoin enantiomer remaining in the reaction mixture.

It has now been found that when the reaction is carried out at a higher pH, for example at a pH of from 7.0 to 9.0, preferably at a pH of from 7.5 to 8.5 , then almost no enzyme inhibition occurs while the undesired enantiomer is racemized. The result is a much higher yield than 50%, which is theoretically the maximum possible yield without racemization. '

The process of the present invention can also be used for cleavage processes in which the DL-carbamoylamino acid is converted into the corresponding amino acid enriched with the D-enantiomer and the unconverted L-N-carbamoylamino acid enriched with the aid of. A microorganism comprising a D-selective hydantoin hydrolysis enzyme and a N-carbamoylamino acid hydrolyzing enzyme. Since this reaction can be carried out at an elevated pHf value (e.g., in the range of 7.5 to 9), there is less loss of L-N-carbamoylamino acid, or a hydantoinase reaction does not occur at a higher pH. ·

IN

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DETAILED DESCRIPTION OF THE INVENTION

The process of the present invention will be explained in more detail below with reference to specific examples, which are illustrative only and not limiting.

Example 1

34 grams of DL-p-hydroxyphenylglycine hydantoin and 34 grams of MgHPCl 3 .3H 2 O were added to 200 ml of water. The pH of the reaction mixture was then adjusted to pH 7.2 with a 5 N sodium hydroxide solution and then started by adding 34 ml of Agrobacterium radiobacter cell suspension. This reaction was carried out under a nitrogen atmosphere at 40 ° C. The pH of the reaction mixture was kept constant at 7.2 by addition of 5 N sodium hydroxide solution. ;

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HPLC analysis confirmed that a conversion of > 99% was achieved after 10 hours of hydrolysis.

Comparative procedure.

'AND

To carry out this comparative procedure, 34 grams of DL-hydroxyphenylglycinhydantoin were hydrolyzed under comparable conditions to that described in Example 1, but in the absence of the phosphate salt. The reaction was started by adding 34 ml of the same Agrobacterium radiobacter cell suspension. This reaction was carried out under a nitrogen atmosphere at 40 ° C. The pH of this reaction mixture was kept constant at 7.2 by automatic titration with 1.3 µM H 3 PO 4 solution. Reaction development was monitored with regular •

After approximately 10 hours, approximately 57. Conversion intervals by HPLC analysis, hydrolysis showed a conversion after 30 hours corresponding to> 99%.

Example 2

DL-p-hydroxyphenylglycine hydantoin was hydrolyzed by adding 122 grams of this compound together with 122 grams of MgHPO 3 · 3H 2 O to 575 milliliters of water. The pH of the mixture was adjusted to 7.2, and nitrogen gas was passed through the mixture for 1 hour and 1 hour. Enzymatic conversion was started by adding 8 ml of Agrobacterium rajobacter cell suspension.

The pH of the reaction mixture was kept constant at 7.2 by addition of sodium hydroxide (25% by weight). The total consumption of sodium hydroxide was 19 grams. After 95 hours of hydrolysis, the conversion was 99.3%.

Comparative procedure

To perform this comparative procedure, p-hydroxyphenylglycine hydantoin (122 grams) was added to 575 ml of water at 40 ° C under comparable conditions as in Example 2.

7.2 with 5 N sodium hydroxide solution was passed through the reaction mixture with nitrogen gas for 1 hour. The reaction was started by adding 30 ml of the same Agrobacterium rajobacter cell suspension. The pH was then maintained at a constant level of 7.2 by the addition of H3PQ4 '(33% by weight) phosphoric acid solution. After 95 hours of hydrolysis, a conversion of 98.7% was found. The conversion was found to be 97.5 hours

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99.3%.

Example 1 a d 3

176 grams of DL-p-hydroxyphenylglycinohydantoin and 176 grams of MgHPO 3 .3H 2 O were added to 510 ml of water at 40 ° C. The reaction mixture was inerted for 1 hour with nitrogen.

Enzymatic conversion was initiated by the addition of 15 ml of Agrobacterium radiobacter cell suspension.

The pH of the reaction mixture was then kept constant at 7.2 using automatic titration with ‘'·

N sodium hydroxide solution. By HPLC analysis was;

it can be found that the conversion after 120 hours of hydrolysis J was 99.8%. ;

Comparative procedure

176 grams of DL-p-hydroxyphenylglycinohydantoin was added to 557 ml of water at 40 ° C and the pH was adjusted to 7.2 with 5 N sodium hydroxide solution. The reaction mixture was inerted for 1 hour with nitrogen, then 56 ml of the same Agrobacterium radiobacter cell suspension were added. The pH of the reaction mixture was then kept constant at 7.2 by automatic titration with 33% by weight phosphoric acid. By HPLC analysis, it was found that the conversion after 146.5 hours of hydrolysis was 96.5% and after 180 hours 99.8%.

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9 9 9

Example 4 j

The reaction mixture obtained in Example 2 was acidified with approximately 130 grams of sulfuric acid (98% by weight) to pH 1, after which the cell debris was removed by microfiltration. The remaining portion was diafiltered. use 100 grams of water. The combined aqueous phases were combined and partially evaporated to a volume of approximately 600 milliliters. Thereafter, approximately 63 grams of sodium hydroxide (concentration; *) was added to the resulting residue.

50% by weight) to a pH of 3.5 and thus formed. ...

D-p-hydroxyphenylglycine was separated and washed several times.

Approximately 53 grams of sodium hydroxide (50% by weight) was added to the filtrate obtained in this step to reach a pH of 8.5 and then separated. (MgNH2 PO4) thus formed. Vs '. ··?' ·. - '

The MgNH2 PO4 was washed twice with 50 ml of water and suspended in water. In the next phase, a mixture of water and ammonia was evaporated under reduced pressure. . ‘

Finally, 101.7 grams of D-p-hydroxyphenylglycine and 118 grams of MgHPO 4. Example 5 f '·.

The pH of the reaction mixture obtained according to Example 2 was adjusted to pH 10 by the addition of approximately 70 grams of sodium hydroxide (50% by weight). The reaction mixture was filtered and the residue was washed twice with 100%.

.. - 13 · f · f f f f f f f f f f f f f f f f f f f f f f f f f f f f Milliliters of water. The filtrate obtained in this step was then passed through a microfiltration system to remove any cell debris. The residue was then washed several times. The permeate was acidified by the addition of approximately 40 grams of sulfuric acid to a pH of 3.5, whereupon D-p-hydroxyphenylglycine crystallized. After separation, washing and drying, 102 g of D-p-hydroxyphenylglycine was obtained.

Example 6

The conversion of DL-p-hydroxyphenylglycinhydantoin to D - (-) - p-hydroxyphenylglycine was carried out at different pH values. ' ₄

- A ^f:.

In this procedure, 34 ‘grams (177 mmol) of DL-p-hydroxyphenylglycine hydantoin and 34 grams of MgHPO ^SSO were added to 200 ml of water. The reaction mixture was adjusted to the desired pH with 5N sodium hydroxide solution and the reaction was started by the addition of 3.0 ml of a cell suspension of Agrobacterium radiobacter. The reaction was then carried out under nitrogen at 40 ° C. The pH of the reaction mixture was then maintained at the desired level, by automatic titration using 5 N sodium hydroxide solution. The results of these experiments are shown in Table 1 below. conversion rates> 99%. .

TABLE 1

PH value Response Time (hours) to Conversion> 99% 7.2 80 7.4 ; 68 7.6 : \ 75. 7.7 .-72 7.8 '7 Z' ⁸⁸ . -

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Example 7 - · i-- V ·· '', v, '·. ·' * 1 ’

According to this procedure he was fp; 40 ° C 30 grams <

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DL-N-carbamoyl-p-hydroxyphenylglycirium and 34 grams of MgHPO4.

added to 200 milliliters of water. The pH of the reaction mixture was then adjusted to 8.0 by the addition of about 15 grams of hydroxide.

of potassium at a concentration of 42%. Subsequently, 13 milliliters of cell suspension was added under a nitrogen atmosphere

Agrobacterium radiobacter. The pH of the reaction mixture was _4% . . , 1.7 maintained at a constant level of 8.0. The composition of this reaction mixture was monitored by HPLC analysis. After a reaction time of 27 hours, the concentration was measured.

D-p-hydroxyphenylglycine, which is 4.3% by weight; 'And the concentration of L-N-carbamoyl-p-hydroxyphenylglycine was'

5.4% by weight. The composition of this reaction mixture remained constant in the subsequent phase. ;

Example 8

Up to 200 milliliters of water was added

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9 9 9 9 9

9 9 9 '9 9 ·

Added 15 grams of DL-valine hydantoin and 20 grams of MgHPO ₄ .3H ₂ 0.>

After adjusting the pH to 8 by addition of 5 N sodium hydroxide solution, the reaction was started by adding 15 ml of a cell suspension of Agrobacterium gladiobacter. _To this reaction was carried out at 40 ° C under nitrogen. The pH was kept constant at 8 by the addition of 5 N sodium hydroxide solution. After 16 hours of hydrolysis, the D-valine concentration was found to be 52 grams / liter, which corresponds to a conversion of > 99% based on DL-valinehydantoin.

Claims (13)

A process for the preparation of a chiral amino acid enriched in D-enantiomer, wherein a mixture of enantiomers of the corresponding N-carbamoylamino acid is contacted with D-carbamoylase, releasing ammonia, characterized in that the ammonia is removed with a phosphate ion salt, hydrogen phosphate ion or a dihydrogen phosphate ion and a divalent metal. The process according to claim 1, wherein the enzymatic decarbamoylation is carried out in the presence of a phosphate ion salt, a phosphate ion or a dihydrogen phosphate ion and a divalent metal. A method according to claim 1, characterized in that:. the reaction mixture is contacted with a divalent metal phosphate, hydrogen phosphate, or dihydrogen phosphate salt in an external circuit after separation of the solid present. 4. A process for the preparation of a chiral amino acid enriched in D-enantiomer, characterized in that the corresponding hydantoin is enzymatically converted with the aid of hydantoinase to the corresponding N-carbamoylamino acid, which is subsequently converted into an amino acid enriched in D-enantiomer using the method according to any of the claims 1-3. The method of claim 4; characterized in that both steps are carried out in a single vessel in the presence of both hydantoinase and D-carbamoylase. ί Method according to claim 4 or 5, characterized in that:. The pH of the reaction mixture is between 7.0 and 9.0. Process according to one of Claims 1 to 6, characterized in that magnesium dibasic phosphate is used as the phosphate ion salt of the divalent metal. The process according to any one of claims 1 to 7, characterized in that the resulting reaction mixture is subsequently subjected to an increase in pH of from 9.5 to 10.5, followed by separation of the solid ammonium phosphate salt formed, the pH of the mother liquor. The caustic is reduced to 3.5 to 4.5 and finally the D-amino acid is separated. Process according to one of Claims 1 to 7, characterized in that the pH of the obtained reaction mixture is subsequently reduced to 0.5 to 1.5, followed by separation of the biomass, then the pH is raised to 3.5 to 4, 5 and the solid D-amino acid is separated. Process according to claim 9, characterized in that the pH of the mother liquor thus obtained is raised to 7 to 10 and the solid ammonium phosphate salt is separated. 11. A process for the recovery of magnesium hydrogen phosphate, characterized in that ammonium phosphate is contacted with a mineral acid at a pH in the range of 4.5 to 6.5, followed by separation of the ammonium salt and the mineral magnesium hydrogen phosphate. The method of claim 11, wherein the pH is maintained in the range of 5.5 to 6. - 18 44 »444 ·· 4ι» 44 4444 »· C 44 0 4 4 4 4> 4 4 4 4 4ι» 4 "• · 4 4 4 4 4 40 _t 444. 4 · 0 • 44 4 44 4 14 4 4 4 4 4 4 4 4 4 '4 4 4 Method according to claim 11 or 12, characterized in that sulfuric acid is used as the mineral acid: