Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing alpha or beta amino acid or substituted amino acid...
Patent
1996-04-08
1998-03-31
Mosher, Mary E.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing alpha or beta amino acid or substituted amino acid...
435122, 435174, 435280, 435227, 4352521, C12P 1304, C12P 1712, C12N 978, C12N 120, C12N 1100
Patent
active
057337543
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
This invention relates to an enzyme and its use, for example in preparing (S)-pipecolic acid.
BACKGROUND OF THE INVENTION
(S)-Pipecolic acid is an intermediate in the preparation of levobupivacaine, the less cardiotoxic enantiomer of the analgesic and anaesthetic agent bupivacaine. Various methods for preparation of this synthon are known.
Ng-Young-Chen et al, J. Org. Chem. 59:2075-81 (1994), describe the resolution of pipecolic acid esters using Aspergillus niger lipase. Low ee (S)-pipecolic acid was produced. An enhancement in enantioselectivity could be obtained through partial purification of the enzyme, but in this case a maximum ee of only 93% was obtained. Use of N-acyl-pipecolic acid ester substrates resulted in no improvement in enantioselectivity.
Toone and Jones, Can. J. Chem. 65:2722-6 (1987), report on the utility of pig liver esterase to carry out similar pipecolic acid ester resolutions ee values were found to be less than 50%.
Mochizuki et al, Agric. Biol. Chem. 52:1113-6 (1988), describe the use of an Alcaligenes strain to selectively metabolise (S)-pipecolic acid to accumulate (S)-.alpha.-aminoadipic acid and leave (R)-pipecolic acid. This is not suitable for (S)-pipecolic acid production and results in loss of a significant proportion of the (S)-.alpha.-aminoadipic acid due to its further metabolism.
An alternative approach has been described by Huh et al, Biosci. Biotech. Biochem. 56:2081-2 (1992), who used D-amino-acid oxidase from porcine kidney to selectively oxidise (R)-pipecolic acid to .DELTA..sup.1 -piperidine-2-carboxylic acid and leave (S)-pipecolic acid as product. The .DELTA..sup.1 -piperidine-2-carboxylate could be recycled to (R),(S)-pipecolic acid in the reaction mixture through the addition of sodium borohydride, but at elevated concentrations this was inhibitory to the oxidase enzyme. Industrial use of this system would additionally be restricted by the cost, poor availability and low stability of the enzyme.
The use of amino acylases to resolve N-acylamino-acids is well established. Verkhovskaya et al, Russian Chemical Reviews 60:1163-79 (1991), review this and other technologies for producing amino-acids. In particular, they disclose the use of acylases from Aspergillus sp., Bacillus sp. or porcine kidney for the resolution primary amino-acids. However, such enzymes do not have activity against secondary N-acylamino-acids.
EP-A-0416282 discloses a N-acylamino-acid acylase, to produce (S)-proline. The addition of substrate during the fermentation was necessary in order to obtain the required activity. N-acyl-(S)-proline (the primary substrate) is expensive and will be consumed during the fermentation. The activity of the enzyme on N-acyl-pipecolic acid was very low, at less than 5% of its activity on N-acetyl-proline, i.e. N-acyl-pipecolic acid compounds are very poor substrates for the enzyme.
SUMMARY OF THE INVENTION
A microbial N-acyl-pipecolic acid acylase has been found, that has good potential for long term re-use, that does not require addition of inducer for expression of activity, and that has activity on N-acyl-pipecolic acid at least comparable to its activity on N-acyl-proline. Its preferred activities are based on Example 6 herein. Specifically, the enzyme preferably has acylase activity capable of hydrolysing N-acetyl-(R,S)-pipecolic acid stereoselectively, to give (S)-pipecolic acid, wherein said activity is greater than that on N-acetyl-S-proline, at pH 7.5, 25.degree. C., and substrate concentration 20 g/l, in 75 mM Tris buffer, and more preferably also has activity on N-acetyl-(R,S)-alanine that is at least 40%, preferably at least 50%, of the activity on N-acetyl-(S)-proline under the foregoing conditions.
In addition, it is surprisingly found that the novel enzyme has activity on primary N-acyl-amino-acids. Thus both aromatic and aliphatic amino-acids such as phenylalanine and alanine may be resolved using the enzyme to give the (S) amino acid with an ee of at least 95%, and often greater than 98%. This broad activity ra
REFERENCES:
patent: 5120652 (1992-06-01), Groeger et al.
patent: 5219741 (1993-06-01), Groeger et al.
K. Mochizuki et al., "Simultaneous Production of D-Pipecolic Acid and L-.alpha.-Aminoadipic Acid from DL-Pipecolic Acid Using A Microoragnism", Agric. Biol. Chem. 52(5): 1113-1116 (1988).
E. Toone et al., "Enzymes in organic synthesis. 40..sup.1 Evaluation of the enantioselectivity of the pig liver esterase catalyzed hydrolyses of recemic piperidine carboxylic acid esters", Can. J. Chem. 65: 2722-2726 (1987).
Chemical Abstracts 110(9): 506 (1989), abstract 73875p.
Lee Caroline Susan
Ricaud Paul Maurice
Wisdom Richard Anthony
Chiroscience Limited
Mosher Mary E.
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