Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing alpha or beta amino acid or substituted amino acid...
Reexamination Certificate
2001-10-11
2004-07-27
Patterson, Jr., Charles L. (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing alpha or beta amino acid or substituted amino acid...
C435S233000, C435S280000
Reexamination Certificate
active
06767725
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to the use of an N-acetylamino acid racemase (AAR) in a process for the racemization of N-carbamoyl amino acids.
2. Description of the Background
Optically pure amino acids are important starting materials for chemical synthesis and for parenteral nutrition. Many methods of preparing optically pure amino acids are known. Enzymatic processes, i.a. are suitable in this respect since, on the one hand, they operate catalytically and, on the other hand, permit the preparation of the amino acids with very high enantiomeric enrichment.
A known enzymatic process starts from racemic hydantoins which are transformed to N-carbamoyl-protected amino acids by means of hydantoinases. These are then converted by carbamoylases to the corresponding amino acids.
The separation of the racemates occurring in this reaction sequence takes place preferably on the basis of the N-carbamoyl-protected amino acids because both L- and D-selective carbamoylases are available (Park et al., Biotechnol. Prog. 2000, 16, 564-570; May et al., Nat Biotechnol. 2000, 18, 317-20: Pietzsch et al., J. Chromatogr. B Biomed. Sci. Appl. 2000, 737, 179-86; Chao et al., Biotechnol. Prog. 1999, 15, 603-7: Wilms et al., J. Biotechnol. 1999, 68, 101-13: Batisse et al., Appl. Environ. Microbiol. 1997, 63, 763-6; Buson et al., FEMS Microbiol. Lett. 1996, 145, 55-62, each of which is incorporated herein by reference).
In order to ensure complete conversion of the hydantoins used to optically pure amino acids, the necessary racemization has taken place hitherto on the basis of hydantoins by chemical or enzymatic means (EP 745678: EP 542098; Scheme 1).
N-acetyl amino acid racemases (AARs) from
Streptomyces atratus
Y-53 (Tokuyama et al., Appl. Microbiol. Biotechnol. 1994, 40, 835-840) and Amycolatopis sp. TS-1-60 (Tokuyama et al., App;. Microbiol. Biotechnol. 1995a, 42, 853-859) and
Amycolatopsis orientalis
sp.
lurida
(DE 19935268) are known. TS-1-60, however, is found to have a very low activity in the case of N-carbamoyl-protected amino acids. Moreover, this enzyme has the disadvantage of a very high metal ion dependence, which appears to be a drawback for the use of this enzyme in an industrial-scale process.
Accordingly, there remains a need for improved methods of racemizing N-carbamoyl amino acids which overcome the disadvantages described above.
SUMMARY OF THE INVENTION
The object of the present invention was, therefore, to show the use of an N-acetyl amino acid racemase for the improved racemization of N-carbamoyl amino acids compared to known methods. The intention was that this racemase might be used advantageously on an industrial scale in a process for the preparation of optically pure amino acid starting from racemic hydantoins.
It was another object of the present invention to provide a process for producing enantiomerically enriched amino acids.
The objects of the present invention, and others, may be accomplished with a method of racemizing N-carbamoyl amino acids, comprising:
contacting an N-carbamoyl amino acid with an effective amount of an N-acetyl amino acid racemase (AAR) from
Amycolatopsis orientalis
subspecies
lurida.
The objects of the present invention may also be accomplished with a method of producing enantiomerically enriched amino acids, comprising:
contacting an N-carbamoyl amino acid with an effective amount of an N-acetyl amino acid racemase (AAR) from
Amycolatopsis orientalis
subspecies
lurida
, and
contacting the racemized N-carbamoyl amino acid with a carbamoylase.
The objects of the present invention may also be accomplished with a method of producing enantiomerically enriched amino acids, comprising:
contacting an a hydantoin with a hydantoinase to produce the corresponding N-carbamoyl amino acid,
contacting an N-carbamoyl amino acid with an effective amount of an N-acetyl amino acid racemase (AAR) from
Amycolatopsis orientalis
subspecies
lurida
to produce a racemized N-carbamoyl amino acid, and
contacting the racemized N-carbamoyl amino acid with a carbamoylase to produce the corresponding amino acid.
A more complete appreciation of the invention and many of the attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the following detailed description below.
DETAILED DESCRIPTION OF THE INVENTION
Due to the fact that an N-acetyl amino acid racemase (AAR) from
Amycolatopsis orientalis
subspecies
lurida
(SEQ ID NO.: 2; the encoding nucleic acid sequence is shown in SEQ ID NO.: 1) is used in a process for the racemization of N-carbamoyl amino acids, and in view of the surprisingly high activity of the AAR used according to the invention compared with TS-1-60 in terms of the racemization of N-carbamoyl amino acids, it is possible to achieve an equilibrium of enantiomers of N-carbamoyl-protected amino acids in an improved process.
This is particularly advantageous in that it is thus possible to establish a further enzymatic step in a process for the preparation of optically pure amino acids which is based on hydantoins (Scheme 2).
In contrast to the enzymatic processes known from the literature and which proceed by way of enzymatic or optionally stressing chemical racemization of hydantoins (Scheme 1), a further advantageous possibility of generating optically pure amino acids from racemic hydantoins has thus been created.
The variant of AAR from
Amycolatopsis o
. sp.
lurida
prepared by recombinant technology according to DE 19935268, incorporated herein by reference, is preferably used for the racemization process. It is known from DE 19935268 that this exhibits relatively little heavy metal ion dependence (particularly with regard to cobalt ions) and has low amino acid inhibition. The generation thereof as a recombinant enzyme is also explained therein.
The process according to the invention, as has been mentioned, is used advantageously in an overall process for the preparation of enantiomerically enriched amino acids or derivatives thereof starting from hydantoins or N-carbamoyl amino acids. In the case of hydantoins, it is preferable to proceed in such a manner that racemic hydantoins are cleaved by hydantoinases into the corresponding racemic N-carbamoyl amino acids and these are then converted by L- or D-specific carbamoylases into the optically active L- or D-amino acids. To ensure that no enrichment of the unconverted enantiomer of an N-carbamoyl amino acid takes place in the reaction mixture, the enantiomers of the N-carbamoyl amino acids are brought into equilibrium by the addition of the AAR according to the invention and it is thus likewise possible to convert the racemic hydantoin wholly to optically pure amino acids.
The process of the present invention is preferably conducted in an enzyme-membrane reactor. Such a reactor is described in, for example, DE 199 10 691.6, incorporated herein by reference.
The enzymes mentioned may be used together or successively in the free form as homogeneously purified compounds or as enzymes prepared by recombinant technology. Moreover, the enzymes may also be used as a constituent of a guest organism (whole-cell catalyst as described in U.S. patent application Ser. No. 09/407,062, incorporated herein by reference) or in conjunction with the digested cell mass of the host organism. It is also possible to use the enzymes in the immobilized form (Bhavender P. Sharma, Lorraine F. Bailey and Ralph A. Messing, “Immobilisierte Biomaterialiern—Techniken and Anwendungen”, Angew. Chem. 1982, 94, 836-852, incorporated herein by reference). Immobilization takes place advantageously by freeze-drying (Dordick et al. J. Am. Chem. Soc. 194, 116, 5009-5010, incorporated herein by reference; Okahata et al. Tetrahedron Lett. 1997, 38, 1971-1974, incorporated herein by reference; Adlercreutz et al. Biocatalysis 1992, 6, 291-305, incorporated herein by reference). Freeze-drying in the presence of surfactant substances such as Aerosol OT or polyvinyl pyrrolidone or polyethylene glycol (PEG) or Brij 52 (diethylene
Bommarius Andreas
Drauz Karlheinz
Kula Maria-Regina
Verseck Stefan
Degussa - AG
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
Patterson Jr. Charles L.
LandOfFree
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