Transaminase biotransformation process

Details Transaminase biotransformation process Transaminase biotransformation process

1997-05-19

2001-03-06

Achutamurthy, Ponnathapu (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...

C435S252100, C435S320100, C435S243000, C435S193000

Reexamination Certificate

active

06197558

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a process for producing amino acids employing aminotransferases. More particularly, this invention relates to a process for making unnatural amino acids such as L-2-aminobutyrate and L-tert-leucine employing an aminotransferase.
2. Related Background Art
The process of the invention improves on prior art processes for making natural and unnatural amino acids using transaminase enzymes. Transaminases have been known in the literature for many years. See, Transaminases, Philipp Christen & David E. Metzler, ed. (1985) (John Wiley and Sons, New York). Briefly, a transaminase reaction requires two substrates, an amino acid and a keto acid. The transaminase catalyzes the exchange of the keto group (C═) from the keto acid and the amino group from the amino acid (—NH
2
). This exchange generates a new amino acid from the keto acid and a new keto acid from the amino acid. Typically only one of the products is desired, generally the new amino acid, and the other is an unwanted by-product.
Used in isolation, the enzyme converts the two substrates to the two products. Theoretically, because the reaction is reversible, it proceeds until it reaches equilibrium producing roughly 50% conversion of the substrate to products.
U.S. Pat. No. 4,518,692 (“Rozzell I”) discloses a method for producing L-amino acids by reacting L-aspartic acid and various 2-keto acids with transaminases. The Rozzell I method uses L-aspartic acid as the amino acid to produce oxaloacetate and describes various methods of decarboxylating oxaloacetate to form pyruvate. However, as will be shown herein, the pyruvate produced in the Rozzell I method can still act as a keto donor in the reverse process to form alanine. Tokarski et al.,
Biotechnology Letters
, Vol. 10 (1) (1988), pp. 7-10, show that alanine acts as a substrate in transaminase reactions. See also,
Transaminases
(1985); and
Amino Acids: Biosynthesis and Genetic Regulation
, Klaus M. Herrmann and Ronald L. Somerville ed. (1983) (Addison-Wesley Publishing, Reading Mass.). Tokarski, et al. studied the use of a transaminase to produce L-2-aminobutyrate from 2-ketobutyrate and alanine. The reference, however, discloses only 25-30% conversion to products, demonstrating that the reverse reaction will prevent attaining even the theoretical limit of 50%. This has long been considered an intrinsic property and a problem of transaminase reactions and is the major reason such enzyme catalyzed reactions have not been exploited more in industrial processes to produce these highly desired amino acid products. The present invention differs from Rozzell I and Tokarski et al. by providing an effective enzymatic method to remove the potential substrates of the reverse reaction from the mixture.
U.S. Pat. No. 4,826,766 (“Rozzell II”), discloses an improved transaminase catalyzed reaction that employs two transaminase enzymes and additional keto acids. In the process, a first transaminase enzyme catalyzes the reaction between a first amino acid and a first keto acid to produce a second amino acid and second keto acid. A second transaminase enzyme then catalyzes a further reaction of the second amino acid and a third keto acid to form the desired amino acid. The two transaminase enzymes are selected such that the first enzyme does not catalyze the second reaction and the second enzyme does not catalyze the first reaction. The Rozzell II method, however, requires additional keto acid and does not disclose the use of acetolactate synthase.
The disclosure of these patents and references are hereby incorporated in their entirety into this specification by reference. Thus, a method to increase the yield of amino acids using transaminase enzymes is desirable.
This invention provides an improved transaminase process, which combines the transaminase enzyme with a second enzyme that eliminates the keto acid produced by the transaminase reaction, preventing the formation of equilibrium, and driving the amino acid producing reaction to completion. The second enzyme catalyzes a reaction which converts the keto acid to a substance that can no longer react with the transaminase. By removing the second keto acid, the second enzyme allows the amino acid producing reaction to proceed to an extent that the desired amino acid product represents approximately 100% of the amino acids produced.
The amino acids produced by this process are useful by themselves, for example as feed additives, flavor enhancers, sweeteners, and nutritional supplements, or can be used as synthetic intermediates to be further reacted to form useful products, in particular pharmaceuticals. Amino acid products of this process are particularly useful as single enantiomer starting materials for producing chiral pharmaceuticals.
SUMMARY OF THE INVENTION
This invention provides a process for making an amino acid which comprises reacting a first amino acid, a first keto acid and a transaminase enzyme under conditions appropriate to produce a second amino acid and pyruvate; and reacting the pyruvate with acetolactate synthase under conditions appropriate to produce a compound that does not react with the transaminase enzyme.
The invention also provides a process for producing an amino acid which comprises:
a) reacting a first amino acid with an enzyme under conditions appropriate to produce a keto acid;
b) reacting the keto acid with a second amino acid and a transaminase enzyme under conditions appropriate to produce a third amino acid and pyruvate; and
c) reacting pyruvate with acetolactate synthase.
The invention further provides a process for making L-2-aminobutyrate comprising:
a) reacting L-threonine with threonine deaminase under conditions appropriate to produce 2-ketobutyrate;
b) reacting the 2-ketobutyrate, L-aspartate and transaminase enzyme under conditions appropriate to produce oxolactate and L-2-aminobutyrate;
c) allowing the oxolactate to form pyruvate;
d) reacting the pyruvate with acetolactate synthase enzyme under conditions appropriate to produce acetolactate;
e) allowing the acetolactate to form acetoin; and
f) separately recovering acetoin and L-2-aminobutyrate.
The invention also provides a process for producing 2-amino butyrate which comprises:
a) reacting L-threonine with a threonine deaminase enzyme under conditions appropriate to produce 2-ketobutyrate;
b) reacting 2-ketobutyrate with an amino acid and transaminase enzyme under conditions appropriate to produce 2-aminobutyrate and pyruvate; and
c) reacting pyruvate with acetolactate synthase enzyme under conditions appropriate to produce a compound that does not react with transaminase enzyme.
The invention also provides a reaction medium comprising a keto acid, an amino acid, transaminase enzyme, and acetolactate synthase enzyme.


REFERENCES:
patent: 4518692 (1985-05-01), Rozzell
patent: 4826766 (1989-05-01), Rozzell
patent: 4880738 (1989-11-01), Rozzell
patent: 5316943 (1994-05-01), Kidman et al.
patent: 5420021 (1995-05-01), Marugg et al.
patent: 500188 A2 (1992-08-01), None
A. Aristidou, Biotechnol Prog., 1995, vol. 11, pp. 475-478.
C. Platteeuw, Applied and Environmental Microbiology, 1995, vol. 61, pp. 3967-3971.
J. Marugg, Applied and Environmental Microbiology, 1994, vol. 60, pp. 1390-1394.
I. Fotheringham, BioChem J., 1986, vol. 234, pp. 593-604.

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