EP1404854A2 - Process for the preparation of enantiomer-enriched amino acids - Google Patents
Process for the preparation of enantiomer-enriched amino acidsInfo
- Publication number
- EP1404854A2 EP1404854A2 EP02711530A EP02711530A EP1404854A2 EP 1404854 A2 EP1404854 A2 EP 1404854A2 EP 02711530 A EP02711530 A EP 02711530A EP 02711530 A EP02711530 A EP 02711530A EP 1404854 A2 EP1404854 A2 EP 1404854A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- phosphate ion
- process according
- enantiomer
- reaction mixture
- amino acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
- 238000000034 method Methods 0.000 title claims abstract description 34
- 150000001413 amino acids Chemical class 0.000 title claims abstract description 13
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims abstract description 49
- 239000011541 reaction mixture Substances 0.000 claims abstract description 29
- 229910021529 ammonia Inorganic materials 0.000 claims abstract description 24
- 239000002253 acid Substances 0.000 claims abstract description 16
- 229940091173 hydantoin Drugs 0.000 claims abstract description 15
- WJRBRSLFGCUECM-UHFFFAOYSA-N hydantoin Chemical compound O=C1CNC(=O)N1 WJRBRSLFGCUECM-UHFFFAOYSA-N 0.000 claims abstract description 14
- 229940085991 phosphate ion Drugs 0.000 claims abstract description 13
- -1 N-carbamoylamino Chemical group 0.000 claims abstract description 11
- 150000003839 salts Chemical class 0.000 claims abstract description 10
- 102100036238 Dihydropyrimidinase Human genes 0.000 claims abstract description 9
- 108091022884 dihydropyrimidinase Proteins 0.000 claims abstract description 9
- 229910052751 metal Inorganic materials 0.000 claims abstract description 9
- 239000002184 metal Substances 0.000 claims abstract description 9
- 239000007787 solid Substances 0.000 claims abstract description 8
- NBIIXXVUZAFLBC-UHFFFAOYSA-L Phosphate ion(2-) Chemical compound OP([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-L 0.000 claims abstract description 7
- NBIIXXVUZAFLBC-UHFFFAOYSA-M dihydrogenphosphate Chemical compound OP(O)([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-M 0.000 claims abstract description 7
- 230000002255 enzymatic effect Effects 0.000 claims abstract description 7
- 239000000203 mixture Substances 0.000 claims abstract description 7
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims abstract description 7
- 238000000926 separation method Methods 0.000 claims abstract description 7
- 108010000622 N-carbamoyl-D-amino acid amidohydrolase Proteins 0.000 claims abstract description 6
- HBAQYPYDRFILMT-UHFFFAOYSA-N 8-[3-(1-cyclopropylpyrazol-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl]-3-methyl-3,8-diazabicyclo[3.2.1]octan-2-one Chemical class C1(CC1)N1N=CC(=C1)C1=NNC2=C1N=C(N=C2)N1C2C(N(CC1CC2)C)=O HBAQYPYDRFILMT-UHFFFAOYSA-N 0.000 claims abstract description 3
- ZRIUUUJAJJNDSS-UHFFFAOYSA-N ammonium phosphates Chemical compound [NH4+].[NH4+].[NH4+].[O-]P([O-])([O-])=O ZRIUUUJAJJNDSS-UHFFFAOYSA-N 0.000 claims description 9
- 150000008574 D-amino acids Chemical class 0.000 claims description 5
- 229910052500 inorganic mineral Inorganic materials 0.000 claims description 5
- 239000011707 mineral Substances 0.000 claims description 5
- MHJAJDCZWVHCPF-UHFFFAOYSA-L dimagnesium phosphate Chemical compound [Mg+2].OP([O-])([O-])=O MHJAJDCZWVHCPF-UHFFFAOYSA-L 0.000 claims description 4
- 239000002028 Biomass Substances 0.000 claims description 3
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 3
- 239000012452 mother liquor Substances 0.000 claims description 3
- 238000011084 recovery Methods 0.000 claims description 3
- 239000001117 sulphuric acid Substances 0.000 claims description 2
- 235000011149 sulphuric acid Nutrition 0.000 claims description 2
- 230000020477 pH reduction Effects 0.000 claims 2
- 239000004254 Ammonium phosphate Substances 0.000 claims 1
- 229910000148 ammonium phosphate Inorganic materials 0.000 claims 1
- 235000019289 ammonium phosphates Nutrition 0.000 claims 1
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 claims 1
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 45
- 238000006243 chemical reaction Methods 0.000 description 35
- 108090000790 Enzymes Proteins 0.000 description 15
- 102000004190 Enzymes Human genes 0.000 description 15
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 13
- 241000589155 Agrobacterium tumefaciens Species 0.000 description 11
- 230000007062 hydrolysis Effects 0.000 description 10
- 238000006460 hydrolysis reaction Methods 0.000 description 10
- 239000006285 cell suspension Substances 0.000 description 9
- LJCWONGJFPCTTL-UHFFFAOYSA-N 4-hydroxyphenylglycine Chemical compound OC(=O)C(N)C1=CC=C(O)C=C1 LJCWONGJFPCTTL-UHFFFAOYSA-N 0.000 description 8
- LJCWONGJFPCTTL-SSDOTTSWSA-N D-4-hydroxyphenylglycine Chemical compound [O-]C(=O)[C@H]([NH3+])C1=CC=C(O)C=C1 LJCWONGJFPCTTL-SSDOTTSWSA-N 0.000 description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 5
- 238000004128 high performance liquid chromatography Methods 0.000 description 5
- 230000005764 inhibitory process Effects 0.000 description 5
- 239000012299 nitrogen atmosphere Substances 0.000 description 5
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Chemical compound CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 4
- 238000006911 enzymatic reaction Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 238000001914 filtration Methods 0.000 description 4
- 239000011777 magnesium Substances 0.000 description 4
- 230000035484 reaction time Effects 0.000 description 4
- 238000004448 titration Methods 0.000 description 4
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 229910052749 magnesium Inorganic materials 0.000 description 3
- 238000001471 micro-filtration Methods 0.000 description 3
- 230000007935 neutral effect Effects 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 102000004879 Racemases and epimerases Human genes 0.000 description 2
- 108090001066 Racemases and epimerases Proteins 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 239000003463 adsorbent Substances 0.000 description 2
- 239000011942 biocatalyst Substances 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 229910001873 dinitrogen Inorganic materials 0.000 description 2
- 238000004821 distillation Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 150000001469 hydantoins Chemical class 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-M hydroxide Chemical compound [OH-] XLYOFNOQVPJJNP-UHFFFAOYSA-M 0.000 description 2
- 238000011065 in-situ storage Methods 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000012465 retentate Substances 0.000 description 2
- 239000002002 slurry Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- GSHIDXLOTQDUAV-ZETCQYMHSA-N (2s)-2-(carbamoylamino)-2-(4-hydroxyphenyl)acetic acid Chemical compound NC(=O)N[C@H](C(O)=O)C1=CC=C(O)C=C1 GSHIDXLOTQDUAV-ZETCQYMHSA-N 0.000 description 1
- 241000590020 Achromobacter Species 0.000 description 1
- 241000186046 Actinomyces Species 0.000 description 1
- 241000589158 Agrobacterium Species 0.000 description 1
- 229940121848 Ammonia scavenger Drugs 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000193764 Brevibacillus brevis Species 0.000 description 1
- 241000186146 Brevibacterium Species 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 1
- 241000186216 Corynebacterium Species 0.000 description 1
- KZSNJWFQEVHDMF-SCSAIBSYSA-N D-valine Chemical compound CC(C)[C@@H](N)C(O)=O KZSNJWFQEVHDMF-SCSAIBSYSA-N 0.000 description 1
- 229930182831 D-valine Natural products 0.000 description 1
- 241001600125 Delftia acidovorans Species 0.000 description 1
- 241000193385 Geobacillus stearothermophilus Species 0.000 description 1
- 241000588748 Klebsiella Species 0.000 description 1
- 241001467578 Microbacterium Species 0.000 description 1
- GSHIDXLOTQDUAV-UHFFFAOYSA-N N-Carbamoyl-2-amino-2-(4-hydroxyphenyl)acetic acid Chemical compound NC(=O)NC(C(O)=O)C1=CC=C(O)C=C1 GSHIDXLOTQDUAV-UHFFFAOYSA-N 0.000 description 1
- BPQQTUXANYXVAA-UHFFFAOYSA-N Orthosilicate Chemical compound [O-][Si]([O-])([O-])[O-] BPQQTUXANYXVAA-UHFFFAOYSA-N 0.000 description 1
- 241001236817 Paecilomyces <Clavicipitaceae> Species 0.000 description 1
- 241000235648 Pichia Species 0.000 description 1
- 241000586779 Protaminobacter Species 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 241000589540 Pseudomonas fluorescens Species 0.000 description 1
- 241000589776 Pseudomonas putida Species 0.000 description 1
- 241000223252 Rhodotorula Species 0.000 description 1
- 241000192023 Sarcina Species 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- 108010046334 Urease Proteins 0.000 description 1
- QCWXUUIWCKQGHC-UHFFFAOYSA-N Zirconium Chemical compound [Zr] QCWXUUIWCKQGHC-UHFFFAOYSA-N 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- MXZRMHIULZDAKC-UHFFFAOYSA-L ammonium magnesium phosphate Chemical compound [NH4+].[Mg+2].[O-]P([O-])([O-])=O MXZRMHIULZDAKC-UHFFFAOYSA-L 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 239000003782 beta lactam antibiotic agent Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 125000001951 carbamoylamino group Chemical group C(N)(=O)N* 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 229910017052 cobalt Inorganic materials 0.000 description 1
- 239000010941 cobalt Substances 0.000 description 1
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000000347 magnesium hydroxide Substances 0.000 description 1
- 229910001862 magnesium hydroxide Inorganic materials 0.000 description 1
- 239000000395 magnesium oxide Substances 0.000 description 1
- CPLXHLVBOLITMK-UHFFFAOYSA-N magnesium oxide Inorganic materials [Mg]=O CPLXHLVBOLITMK-UHFFFAOYSA-N 0.000 description 1
- AXZKOIWUVFPNLO-UHFFFAOYSA-N magnesium;oxygen(2-) Chemical compound [O-2].[Mg+2] AXZKOIWUVFPNLO-UHFFFAOYSA-N 0.000 description 1
- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 150000002739 metals Chemical class 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 229910000069 nitrogen hydride Inorganic materials 0.000 description 1
- 239000000813 peptide hormone Substances 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000009790 rate-determining step (RDS) Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 229910052707 ruthenium Inorganic materials 0.000 description 1
- 230000002000 scavenging effect Effects 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 229910052726 zirconium Inorganic materials 0.000 description 1
- 239000002132 β-lactam antibiotic Substances 0.000 description 1
- 229940124586 β-lactam antibiotics Drugs 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
- C12P41/003—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions
- C12P41/005—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions by esterification of carboxylic acid groups in the enantiomers or the inverse reaction
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
Definitions
- 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 ⁇ -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.
- carbamoylase also called N-carbamoyl-D-amino acid amidohydrolase
- the concentration at which ammonia-caused inhibition occurs is reported (Olivieri et. al. (1981) Biotechnology and Bioengineering, vol.
- the ammonia formed is removed by distillation.
- this process for the removal of ammonia is effective only when the reaction mixture has a high pH.
- the carbamoylase enzyme is not active.
- 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.
- 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.
- the enzymatic reaction can be carried out (for example) in the presence of a phosphate salt.
- 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 ⁇ molar).
- Suitable bivalent metal ions are magnesium, cobalt, calcium, manganese, zirconium or ruthenium ions.
- magnesium monohydrogen phosphate MgHPO 4
- MgHPO 4 fits 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 MgHPO 4
- MgHPO 4 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.
- suitable enzymes 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.
- Pseudomonas in particular Pseudomonas fluorescens, putida or desmolytica
- Achromobacter Corynebacterium
- 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.
- 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.
- the D-amino acid can be separated, for example by filtration or centrifugation.
- 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.
- the biomass is subsequently removed, for example by means of microfiltration or ultrafiltration.
- 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.
- 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.
- hydantoin route 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.
- 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.
- 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.
- a hydrolysis of DL-p-hydroxyphenylglycine hydantoin was carried out by adding 122 g of this compound with 122 g MgHPO 4 -3H 2 O to 575 ml water.
- the enzymatic conversion was started by addition of 8 ml Agrobacterium radiobacter cell suspension.
- the MgNH 4 PO 4 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.
- 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 H 2 SO 4 to 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 VIII 15 g DL-valine hydantoin and 20 g MgHPO 4 -3H 2 O was added to
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- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
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- Bioinformatics & Cheminformatics (AREA)
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
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
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.
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NL1017250A NL1017250C1 (en) | 2001-01-31 | 2001-01-31 | Process for the preparation of enantiomerically enriched amino acids. |
PCT/NL2002/000072 WO2002061107A2 (en) | 2001-01-31 | 2002-01-31 | Process for the preparation of enantiomer-enriched amino acids |
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JP (1) | JP2004521623A (en) |
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CN (1) | CN1520460A (en) |
AU (1) | AU2002230274A1 (en) |
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KR100600698B1 (en) * | 2004-08-26 | 2006-07-14 | 삼성전자주식회사 | Remote control device for controlling video player and video player and their channel switching method |
EP2508615A4 (en) | 2009-12-04 | 2013-07-10 | Mitsubishi Gas Chemical Co | Process for production of optically active amino acid or optically active amino acid amide |
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DE3732896A1 (en) * | 1986-11-07 | 1988-08-25 | Schulze Rettmer Rainer | Process for eliminating ammonia and phosphate from waste water and process water |
DE4040067C2 (en) * | 1990-12-14 | 1994-04-07 | Nalco Chemie Gmbh Deutsche | Process for the removal and recovery of ammonium contents from process and waste water |
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