Biotechnical production process of piperazine...

Details Biotechnical production process of piperazine... Biotechnical production process of piperazine...

1997-11-10

2001-04-10

Marx, Irene (Department: 1651)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C435S252100, C435S280000

Reexamination Certificate

active

06214604

ABSTRACT:

The invention relates to novel microorganisms which are capable of converting, in optionally substituted (RS)-&agr;-piperazinecarboxamides, of the formula
the optionally substituted R-&agr;-piperazinecarboxamide into the corresponding optionally substituted R-&agr;-piperazinecarboxylic acid, of the formula
The microorganisms are capable of utilizing optionally substituted &agr;-piperazinecarboxamides of the above formula I, in the form of their racemate or their optically active isomers, in particular R-&agr;-piperazinecarboxamides, as the only nitrogen source. These microorganisms, or their cell-free enzymes, are employed in a novel process for the preparation of optionally substituted R-&agr;-piperazinecarboxylic acids (formula II) and/or for the preparation of optionally substituted S-&agr;-piperazinecarboxamides, of the formula
R-&agr;-Piperazinecarboxylic acid, of the formula II, is an important intermediate for the preparation of (R)-2-carboxy-4-(3-phosphonopropyl)piperazine, which is a selective antagonist of N-methyl-D-aspartate (Synlett, 1996, 143-144).
A microbiological process for the preparation of R-&agr;-piperazinecarboxylic acid has hitherto not been described in the literature.
It is an object of the present invention to provide a simple, technically feasible biotechnological process for the preparation of optically pure R-&agr;-piperazinecarboxylic acids which simultaneously also allows S-&agr;-piperazinecarboxamide to be isolated in high purity.
This object is achieved by the microorganisms according to patent claim
1
and by the process according to patent claim
3
.
The microorganisms according to the invention can be isolated from soil samples, sludge or wastewater with the aid of customary microbiological techniques. According to the invention, these microorganisms are isolated in such a way that they are
a) grown in the customary manner in a medium with an optionally substituted &agr;-piperazinecarboxamide (formula I) in the form of its racemate or its optically active isomers as the only nitrogen source, preferably with an R-&agr;-piperazinecarboxamide as the only nitrogen source, and with a suitable carbon source;
b) then, those which are stable and capable of converting, in (RS)-&agr;-piperazinecarboxamides (formula I), the R-&agr;-piperazinecarboxamide into the corresponding R-&agr;-piperazinecarboxylic acid (formula II) are selected from the culture obtained by growing.
Accordingly, all microorganisms which specifically contain the R-piperazinecarboxamidases may be employed.
Examples of carbon sources which the microorganisms can utilize as growth substrates are sugars, sugar alcohols or carboxylic acids. Sugars which can be used are hexoses such as, for example, glucose, or pentoses. Carboxylic acids which can be used are di- or tricarboxylic acids or their salts such as, for example, citric acid or succinate. A sugar alcohol which may be used is, for example, glycerol. A sugar alcohol such as glycerol is preferably employed as the carbon source.
The selection and growth media which can be used are those conventionally used in expert circles, such as, for example, the mineral salt medium of Kulla et al. (Arch. Microbiol., 135, 1-7, 1983) or the medium described in Table 1. The medium described in Table 1 is preferably used.
It is expedient to induce the effective enzymes of the microorganisms during the growth and selection stage. Piperazinecarboxamide can be employed as the enzyme inductor.
The growth and selection stages are expediently carried out at a temperature of from 15 to 55° C., preferably from 20 to 45° C., and a pH of between pH 5 and pH 11, preferably between pH 6 and pH 10.
Preferred microorganisms with specific R-piperazinecarboxamidase activity are microorganisms of the genus Burkholderia, as well as their functionally equivalent variants and mutants. Especially preferred microorganisms are those of the genus Burkholderia, as deposited on Apr. 20, 1995 at the Deutschen Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig in accordance with the Budapest Treaty as DSM 9925, and their functionally equivalent variants and mutants.
Taxonomic characteristics of microorganisms of the genus Burkholderia (DSM 9925)
Cell shape
rods
Width &mgr;m
0.7-0.8
Length &mgr;m
1.5-3.5
Gram reaction
−
Lysis by 3% KOH
+
Aminopeptidase (Cerny)
+
Spores
−
Oxidase
+
Catalase
+
Pigments
yellow
Growth
−
anaerobic
PNPG
+
ADH
−
Urease
−
Hydrolysis of gelatine
−
Substrate utilization
Adipate
+
Citrate
little
Malate
+
Glucose
−
Adonitol
−
Mannitol
−
Suberate
+
Acetamide
+
2,3-Butylene glycol
+
m-Hydroxybenzoate
+
&agr;-Amylamine
+
Tryptamine
−
Abbreviations:
PNPG: p-nitrophenyl-galactosidase
ADH: alcohol dehydrogenase
“Functionally equivalent variants and mutants” are to be understood as meaning microorganisms which have essentially the same characteristics and functions as the original microorganisms. Such variants and mutants can be formed accidentally, for example by UV irradiation.
The process according to the invention for the preparation of optionally substituted R-&agr;-piperazinecarboxylic acid, of the formula
and/or of optionally substituted S-&agr;-piperazinecarboxamides, of the formula
is carried out in such a way that, in the optionally substituted (RS)-&agr;-piperazinecarboxamide, of the formula
the corresponding R-&agr;-piperazinecarboxamide is converted into the corresponding R-&agr;-piperazinecarboxylic acid by means of the specific microorganisms which have already been described or by means of cell-free enzymes from these microorganisms, and isolated, the biotransformation not only giving R-&agr;-piperazinecarboxylic acid, but also S-&agr;-piperazinecarboxamide, which, if appropriate, is isolated.
The starting materials, the (RS)-&agr;-piperazinecarboxamides of the formula
which are optionally substituted can be obtained from the corresponding aromatic amides by hydrogenation methods conventionally used in the art.
The piperazinecarboxamides of the formula I which are employed can be substituted or unsubstituted. Representatives of substituted piperazinecarboxamides may be C
1
-C
4
-alkyl-substituted, such as, for example, 4-methylpiperazinecarboxamide, H
2
N—CH
2
-substituted, such as, for example, 4-aminomethylpiperazinecarboxamide or acyl-substituted, such as 4-acetylpiperazinecarboxamide. Piperazinecarboxamide or 4-methylpiperazinecarboxamide are preferably used.
The enzymes for the cell-free system can be obtained by rupturing the microorganisms by methods conventionally used in the art. Methods which can be used are, for example, ultrasonic, French press or lysozyme method. These cell-free enzymes can also be immobilized on a suitable support material.
Especially suitable for the process are the above-described specific microorganisms of the genus Burkholderia (DSM 9925), and their functionally equivalent variants and mutants.
After conventionally growing the microorganisms, biotransformation can be effected on dormant cells (non-growing cells which no longer require a carbon and energy source) or on growing cells.
Media which can be used for the process with dormant cells are those conventionally used in the art, such as, for example, the above-described mineral salt medium of Kulla et al., 1983 (ibid), low-molecular-weight phosphate buffers, HEPES buffers, or the medium described in Table 1. A medium which is used for the process with growing cells is normally one which comprises a carbon and nitrogen source, such as, for example, commercially available media or the medium of Table 1. The process is preferably carried out in the medium of Table 1.
Biotransformation is expediently effected with a single or continuous addition of such an amount of (RS)-&agr;-piperazinecarboxamide that the concentration of (RS)-&agr;-piperazinecarboxamide does not exceed 20% by weight, preferably 10% by weight.
The pH of the medium can be in a range of from pH to pH 11, preferably from pH

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