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  • 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
  • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
• • 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
• • 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
  • C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
• • 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
  • C12P13/12—Methionine; Cysteine; Cystine
• • 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
  • C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
  • C12P13/222—Phenylalanine
• • 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
  • C12P41/006—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by reactions involving C-N bonds, e.g. nitriles, amides, hydantoins, carbamates, lactames, transamination reactions, or keto group formation from racemic mixtures
  • C12P41/009—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by reactions involving C-N bonds, e.g. nitriles, amides, hydantoins, carbamates, lactames, transamination reactions, or keto group formation from racemic mixtures by reactions involving hydantoins or carbamoylamino compounds

Definitions

• the invention relates to a process for the preparation of an enantiomerically enriched D- ⁇ -amino acid in a one pot reaction in the presence of a hydantoinase and a D-carbamoylase.
• D- ⁇ -amino acids can be prepared enzymatically from a mixture of the corresponding D- and L-5-substituted hydantoin. Such a process is known from EP 0 775 748.
• a mixture of D- and L-5-substituted hydantoin is enantioselectively converted into the corresponding D- ⁇ -amino acid in a one pot reaction in the presence of a D-hydantoinase and a D-carbamoylase.
• Enantiomerically enriched ⁇ -amino acids are important building blocks for biologically active preparations such as, for example, ⁇ -lactam antibiotics, peptide hormones and pesticides.
• a drawback of this process is that the starting material for the preparation of the enantiomerically enriched ⁇ -amino acid, the corresponding 5- substituted hydantoin, is usually difficult to handle due to the poor water solubility of most 5-substituted hydantoins.
• the invention aims to provide a simple process for the preparation of a D- ⁇ -amino acid in which use can be made of a starting material that it easier to handle than a 5-substituted hydantoin. According to the invention this is achieved through conversion of an N-carbamoyl-L- ⁇ -amino acid into the corresponding D- ⁇ - amino acid.
• N-carbamoyl-L- ⁇ -amino acids can easily be prepared from the corresponding L- ⁇ -amino acids the invention also provides a process by means of which D- ⁇ -amino acids can be prepared rapidly and in a high yield from the corresponding L- ⁇ -amino acids.
• L- ⁇ -amino acids are usually cheap and widely available.
• the process according to the invention is eminently suited for preparing D- ⁇ -amino acids from L- ⁇ -amino acids which, when they are chemically converted into the corresponding hydantoins, yield less stable hydantoins (as a result of which a great deal of by-product formation takes place on chemical conversion of these L- ⁇ -amino acids into the corresponding hydantoins).
• ⁇ -amino acids are serine, asparagine, glutamine, lysine, threonine and cysteine.
• carbamoylase is known from the literature.
• the expert understands carbamoylase to be an enzyme with carbamoylase activity, that is, the ability to catalyze the conversion of N-carbamoyl- ⁇ -amino acid into the corresponding ⁇ -amino acid.
• the expert understands D-carbamoylase to be an enzyme that preferably catalyzes the conversion of N-carbamoyl-D- ⁇ -amino acid into the corresponding D- ⁇ -amino acid.
• the D-carbamoylase involved in the process for the preparation of a D- ⁇ -amino acid according to the invention preferably has a selectivity of at least 90%, more in particular at least 95%, even more in particular at least 98% and most particularly at least 99%.
• a 90% selectivity of the D-carbamoylase is understood to mean that the D-carbamoylase converts the N- carbamoyl-(D,L)- ⁇ -amino acid into 90% D- ⁇ -amino acid and 10% L- ⁇ -amino acid at an overall conversion of 50%, which corresponds to an enantiomeric excess (e.e.) of 80% of the D- ⁇ -amino acid at 50% conversion.
• hydantoinase is known from the literature. The expert understands hydantoinase to be an enzyme with hydantoinase activity, that is, the ability to catalyze the reaction of 5-substituted hydantoin to form the corresponding N- carbamoyl- ⁇ -amino acid.
• the hydantoinase activity that is required for the process according to the invention may be provided by one or by more hydantoinases.
• the enantioselectivity of the hydantoinase(s) involved in the conversion of N-carbamoyl-L- ⁇ -amino acid into the corresponding D- ⁇ -amino acid is not very critical.
• a D-hydantoinase for example, a D-hydantoinase, an L-hydantoinase or a non enantioselective hydantoinase may be employed as well as combinations thereof.
• the expert understands a D-hydantoinase to be a hydantoinase that preferably catalyzes the reversible hydrolysis of a 5- substituted D-hydantoin into the corresponding N-carbamoyl-D- ⁇ -amino acid.
• D-hydantoinase can be employed in the process of the invention as it is generally assumed that D-hydantoinases are strictly D-selective (Drauz, K., Waldmann, H, Enzyme catalysis in organic synthesis - a comprehensive handbook, second, completely revised and enlarged edition, volume III, Wiley-VCH Verlag GmbH, Weinheim 2002) and therefore the person skilled in the art would not expect that N- carbamoyl-L- ⁇ -amino acid can be converted by a D-hydantoinase.
• the conversion of N-carbamoyl-L- ⁇ -amino acid into the corresponding D- ⁇ -amino acid according to the invention is carried out in the presence of a hydantoin racemase.
• Hydantoin racemases are known from the literature (for example from WO 01/23582 A1). The expert understands a hydantoin racemase to be an enzyme with hydantoin racemase activity, that is, the ability to catalyze the racemization of the L-5-substituted hydantoin (or D-5 substituted hydantoin).
• N-carbamoyl-L- ⁇ -amino acid into the corresponding D- ⁇ -amino acid according to the invention is carried out at a pH between 5 and 10. In particular, the conversion is carried out at a pH between 6.0 and 7.5.
• the temperature of the conversion of N-carbamoyl-L- ⁇ -amino acid into the corresponding D- ⁇ -amino acid is preferably chosen between 0 and 80°C, in particular between 10 and 50°C, more in particular between 35 and 45°C.
• the reaction conditions in the process according to the invention are chosen such that the formed enantiomerically enriched D- ⁇ -amino acid crystallizes out, facilitating the recovery of the enantiomerically enriched D- ⁇ -amino acid.
• the process for converting N-carbamoyl-L- ⁇ -amino acid into the corresponding D- ⁇ -amino acid is used in the preparation of a D- ⁇ -amino acid from the corresponding L- ⁇ -amino acid.
• Use is made of the same process as described above, to which an extra step is added, namely the preparation of N-carbamoyl-L- ⁇ - amino acid from the corresponding L- ⁇ -amino acid.
• N-carbamoyl- ⁇ - amino acid from the corresponding ⁇ -amino acid can for example be effected chemically by contacting the ⁇ -amino acid with, for example, MOCN, where M represents alkali metal, preferably potassium or sodium in the presence of water.
• MOCN alkali metal
• the temperature used is not critical: the reaction preferably takes place at temperatures between about -5 and 100°C, preferably at temperatures between 0 and 80 C C; the pH of the chemical reaction is preferably chosen between 8 and 11 , more in particular between 9 and 10.
• the preparation of D- ⁇ -amino acid from the corresponding L- ⁇ -amino acid via the corresponding N-carbamoyl-L- ⁇ -amino acid is carried out in a single vessel.
• This has the advantage that no intermediate recovery of the N- carbamoyl- ⁇ -amino acid is needed.
• the invention is in no way limited by the form in which the enzymes are used for the present invention.
• the different enzymes may each independently be present in a certain form.
• the enzymes may for example be present as a crude enzyme solution or as purified enzyme.
• the enzymes may for example also be present in (permeabilized and/or immobilized) cells that naturally or through genetic modification possess the desired enzymes/enzyme activity, or in a lysate of cells with such activity.
• the enzymes can for example also be used in immobilized form or in chemically modified form. It will be clear to the average person skilled in the art that use can also be made of mutants of naturally occurring (wild type) enzymes with hydantoinase and/or enantioselective carbamoylase and/or hydantoin racemase activity in the process according to the invention.
• Mutants of wild-type enzymes can for example be made by modifying the DNA encoding the wild type enzymes using the mutagenesis techniques known to the person skilled in the art (random mutagenesis, site-directed mutagenesis, directed evolution, gene shuffling, etc.) so that the DNA encodes an enzyme that differs by at least one amino acid from the wild type enzyme and by effecting the expression of the thus modified DNA in a suitable (host) cell.
• Suitable hydantoinases and/or D-carbamoylases and/or hydantoin racemases for the process according to the present invention can be present in, or originate from, for example the microorganisms that usually also supply the enzymes for the hydantoinase-carbamoylase processes, for example the microorganisms of the following genera: Pseudomonas, preferably Pseudomonas sp., more in particular Pseudomonas sp.
• FERM BP 1900 Hansenula, Agrobacterium, preferably Agrobacterium sp. or Agrobacterium radiobacter. more in particular Agrobacterium sp.
• Aerobacter preferably Aerobacter cloacae, more in particular Aerobacter cloacae IAM 1221 , Aeromonas, Bacillus, preferably Bacillus macroides, more in particular Bacillus macroides ATCC 12905. Brevibacterium. Flavobacterium, Serratia, Micrococcus, Arthrobacter, preferably Arthrobacter aurescens. more in particular Arthrobacter aurescens DSM 3747 Nocardia, Corvnebacterium, Mvcobacterium. Actinoplanes, Streptomvces or Paracoccus.
• hydantoinases and/or D-carbamoylases and/or hydantoin racemases may vary dependent on the specific substrate/desired D- ⁇ -amino acid and can easily be determined by the person skilled in the art.
• one or more of the enzymes used in the conversion of N-carbamoyl-L- ⁇ -amino acid into the corresponding D- ⁇ -amino acid according to the invention originate from Agrobacterium radiobacter or Arthrobacter aurescens.
• the invention can be used in the preparation of both proteinogenic and non-proteinogenic D-enantiomers of ⁇ -amino acids having the formula R-CH(NH 2 )- COOH, where R represents an amino acid restgroup, for example a substituted or non- substituted alkyl group with for example 1-20 C atoms or a substituted or a non- substituted (hetero)aryl group with for example 1-20 C atoms.
• Examples of ⁇ -amino acids having the formula R-CH(NH 2 )-COOH are: alanine, valine, leucine, isoleucine, serine, threonine, methionine, cysteine, asparagine, glutamine, tyrosine, tryptophan, aspartic acid, glutamic acid, histidine, lysine, arginine, citrulline, phenylalanine, 3- fluorophenylalanine.
• the invention is used in the preparation of the D- enantiomers of methionine, leucine, cysteine, threonine or phenylalanine.
• Figure 1 shows the conversion of L- ⁇ -amino acid to D- ⁇ -amino acid; the compounds 1, 2 and 3 represent compounds with the L-configuration and the compounds 4, 5, and 6 compounds with the D-configuration.
• R represents an amino acid restgroup as defined above.
• N-carbamoyl-L- ⁇ -amino acid The conversion of N-carbamoyl-L- ⁇ -amino acid from the corresponding L- ⁇ -amino acid is shown in the reaction from 1 to 2.
• a possible route for the conversion of N-carbamoyl-L- ⁇ -amino acid into the corresponding D- ⁇ -amino acid is shown in the reactions from 2 to 6.
• the N-carbamoyl-L- ⁇ -amino acid (2) is converted into the corresponding L-5-substituted hydantoin (3) in the presence of a hydantoinase and a D-carbamoylase.
• the L-5-substituted hydantoin is then racemized in situ.
• Racemization of L-5-substituted hydantoin may occur spontaneously, but can also be effected for example by means of heating or by using a hydantoin racemase.
• D-5 substituted hydantoin present in the mixture of enantiomers of 5-substituted hydantoin (4) formed is subsequently converted into the corresponding N-carbamoyl-D- ⁇ -amino acid (5), after which the N-carbamoyl-D- ⁇ -amino acid is converted to form the corresponding D- ⁇ -amino acid (6), use being made of the presence of a hydantoinase and a D-carbamoylase.
• N-carbamoyl-L-leucine was added to 100 ml water, after which the pH was adjusted to 7.2 by means of 10 N NaOH. The volume was then made up to 150 ml with water. This mixture was heated to 40°C. Subsequently, under a nitrogen atmosphere, the enzymatic reaction was started by the addition of 18 g of an Agrobacterium radiobacter cell suspension. The pH of the reaction mixture was kept constant at pH 7.2 by means of titration with 3 mol/l H 3 PO 4 . The composition of the reaction mixture was monitored by means of HPLC analysis.
• a slurry of 17.9 g (0.093 mol) N-carbamoyl-D,L-methionine in 115 ml water was transferred to a double-walled glass reactor.
• the mixture was heated to 40°C and its pH was adjusted to 7.2 by means of 10 N NaOH, after which the mixture was made up to a volume of 170 ml.
• 12 g of an Agrobacterium radiobacter cell suspension was added, after which the pH of the reaction mixture was kept constant at pH 7.2 by addition of 3 mol/l H 3 PO .
• the reaction was carried out under a nitrogen atmosphere.
• the composition of the reaction mixture was analyzed by means of HPLC analysis. After a reaction time of about 29 hours a conversion of > 88% on the basis of N-carbamoyl-D,L-methionine was reached. The enantiomeric excess of the formed D- methionine was > 96%.
• N-carbamoyl-L-leucine was added to 100 ml water, after which the pH was adjusted to 7.2 by means of 10 N NaOH. The volume was then made up to 170 ml with water. This mixture was heated to 40°C. Subsequently, under a nitrogen atmosphere, the enzymatic reaction was started by the addition of 14.2 g of an Agrobacterium radiobacter cell suspension. The pH of the reaction mixture was kept constant at pH 7.2 by means of titration with 3 mol/l H 3 PO 4 . The composition of the reaction mixture was monitored by means of HPLC analysis. After a reaction time of 44 hours the reaction was stopped.
• the composition of the reaction mixture was analyzed by means of HPLC analysis. After a reaction time of 42 hours a D-leucine concentration of 67 g/l was measured, which corresponds to a conversion of > 95% on the basis of the N- carbamoyl-L-leucine. The enantiomeric excess of the formed D-leucine was > 98%.
• N-carbamoyl-L-amino acid in case of N- carbamoyl-L-cysteine 18.4 g (80 mmol) and in case of N-carbamoyl-L-threonine 17.8g (110 mmol) was added.
• the pH of the mixture was brought to 7.2 using 5 mol/L NaOH, after which the volume was adjusted to 150 mL.
• the mixture was transferred to a thermostated reaction vessel and the temperature was adjusted to 40°C.
• the enzymatic reaction was started by adding 22 g of an Agrobacterium radiobacter celsuspension.
• Example VI A. Construction of plasmid pET101/D-TOPOhyuH
• A. aurescens DSM 3747 was cultivated at 28°C in K2 medium (4 g bacto peptone, 4 g yeast extract, 7.5 g glycin, 2 g KH 2 PO , 3.62 g K 2 HPO 4 , in H 2 O to 1 liter and adjusted to pH 7.0). 10 ml from a preculture was added to 75 ml K2 medium and incubated at 28°C. After 3 hours of incubation, 75 ⁇ l lysozyme (100 mg/ml) and 1.5 ml carbenicillin (50 mg/ml) were added and incubation was continued for another hour at 28°C.
• K2 medium 4 g bacto peptone, 4 g yeast extract, 7.5 g glycin, 2 g KH 2 PO , 3.62 g K 2 HPO 4 , in H 2 O to 1 liter and adjusted to pH 7.0. 10 ml from a preculture was added to 75 ml K2 medium and in
• the cells were isolated by centrifugation and resuspended in 2.5 ml solution consisting of 125 ⁇ l 1 M Tris-HCI, pH 8, 250 ⁇ l 0.5 M EDTA pH 8 and 2.125 ml Milli-Q. Then, 50 ⁇ l lysozyme (100 mg/ml) and 20 ⁇ l proteinase K (20 mg/ml) were added. This suspension was incubated at 37°C for 30 minutes. After adding 3 ml of Nuclei Lysis Solution of Promega (Leiden, The Netherlands), the solution was incubated again for 15 minutes at 80°C.
• a 1377 bp fragment comprising the open reading frame (ORF) for hydantoinase HyuH was amplified by PCR from the chromosomal DNA from Arthrobacter aurescens DSM 3747 (nucleotides 4651-6027 of accession number AF146701) using the following primers: ⁇ yuH-forward:
• the start-codon was changed from GTG into ATG for better expression in E. coli.
• the Platinum Pfx DNA Polymerase of Invitrogen was used to amplify the ORF.
• the 5 pET101/D-TOPOHyuH plasmids were transformed to E. coli BL21 STAR (DE3) One Shot cells (Invitrogen).
• the transformants were cultivated at 28°C in LB medium containing 100 mg/l carbenicillin and induced with 0.5 mM IPTG (final concentration) according to Invitrogen's protocol. After overnight incubation, the cells were harvested by centrifugation and washed with 0.2 M Tris-HCI pH 7.
• the assay mixture of 10 ml contained 0.1 M Tris-HCI buffer pH 8.5 with a) 4 mM phenylalanine hydantoin, or b) 1.8 mM tryptophan hydantoin.
• the assay was performed at 37°C and started by the addition of 0.75 ml cell free extract. Several samples of 0.5 ml were taken in time and the reaction was stopped by adding 5 ⁇ l 85% phosphoric acid to these samples. Next they were analyzed by HPLC and UV detection at a wavelength of 220 nm.
• a Nucleosil-120-5 C18 column (50x4 mm, 5 ⁇ from Macherey-Nagel, D ⁇ ren, Germany) was used. The column was eluted with eluent A (50mM H 3 PO 4 pH 2.7) and eluent B (50 v/v% eluent A and 50 v/v% acetonitril).
• ⁇ /-carbamoyl-phenylalanine or ⁇ /-carbamoyl-tryptophan was produced within 1 hour with cell free extracts derived from all 5 clones of £ co/ pET101 /D-TOPOhyuH tested.
• a 45 mL solution containing 1.8 g (8.6 mmol) N-carbamoyl-L- phenylalanine was brought to pH 7.2 using 5 mol/L NaOH.
• the reaction was thermostated at 40°C.
• the enzymatic reaction was started by adding 2 mL of an Agrobacterium radiobacter celsuspension and 2 mL of a cellfree extract from the £ co/ ET101/D-TOPOhyuH L-hydantoinase clone, which contained the L- hydantoinase from Arthrobacter aurescens DSM 3747.
• the pH of the reaction was kept at pH 7.2.

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