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
2001-03-08
2002-01-15
Lilling, Herbert J. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing alpha or beta amino acid or substituted amino acid...
C435S170000, C435S822000, C435S873000
Reexamination Certificate
active
06338957
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for preparing halo-L-tryptophan from haloindole using a microorganism.
2. Discussion of the Background
Halo-L-tryptophan, for example 5-chloro-L-tryptophan, is useful as a raw material for pharmaceutical products and as a synthetic intermediate for pharmaceutical products.
Methods for producing L-tryptophan from indole using microorganisms include a method using
Escherichia coli
(French Patent No. 1207437) and a method using a microorganism belonging to the genera
Proteus, Erwinia
, Pseudomonas or Aerobacter (Japanese Patent Publication No. 46348/1972). Methods for producing 5-hydroxy-L-tryptophan from 5-hydroxyindole include a method using a microorganism belonging to the genera Proteus, Erwinia, Pseudomonas or Aerobacter (Japanese Patent Publication No. 46348/1972) as well as methods using microorganisms such as
Bacillus subtilis, Corynebacterium hydrocarboclustus, Arthrobacter paraffinens, Micrococcus ureae, Brevibacterium ketoglutamicum, Hansenula anomala
and
Candida tropicalis
(Japanese Patent Publication No. 46349/1972). Additionally, a method for producing 5-amino-L-tryptophan from 5-aminoindole or for producing 5-methyl-L-tryptophan from 5-methylindole is also known (Japanese Patent Publication No. 9760/1977).
Generally, optically active halo-L-tryptophan can be produced by the combination of a chemical synthesis of N-acetyl-halo-DL-tryptophan and the optical resolution of halo-L-tryptophan by aminoacylase, that is, preparing halo-DL-tryptophan by a chemical synthetic method and subsequently acetylating halo-DL-tryptophan into N-acetylhalo-DL-tryptophan, which is further subjected to an optical resolution method with aminoacylase. However, the method requires complicated processes and the presence of residual N-acetyl-halo-D-tryptophan is disadvantageous.
No method is known for producing halo-L-tryptophan from haloindole. Accordingly, there remains a need for an method for producing halo-L-tryptophan from haloindole on an industrially efficient scale which overcomes the disadvantages of the known methods for preparing halo-L-tryptophan.
SUMMARY OF THE INVENTION
The present invention relates to a method for preparing a halo-L-tryptophan using a microorganism.
In particular, the present invention relates to a method for preparing halo-L-tryptophan comprising contacting a haloindole with a microorganism capable of producing halo-L-tryptophan from (a) a mixture comprising haloindole, pyruvic acid and ammonia; or (b) a mixture comprising haloindole and a source of pyruvic acid and ammonia.
In another embodiment, the present invention relates to a method for preparing of halo-L-tryptophan wherein L-tryptophan or a surfactant are also present in a culture medium.
The present inventor has considered that pyridoxal-5′-phosphate is the coenzyme of the enzyme that is involved in the present invention. The inventor have also considered that the molecule of pyridoxal-5′-phosphate might be removed from the enzyme during the cell collection. Therefore, the inventor has added pyridoxal-5′-phosphate into the reaction mixture to supplement the coenzyme. However, in another experiment, the inventor has found that supplementation of pyridoxal-5′-phosphate is not necessary for the present invention.
L-tryptophan is added into the culture medium. The inventor has found that the enzyme is induced by L-trypotphan and added it into the culture medium, not the reaction mixture. Surfactant is also added into the culture medium. The inventor has found that the enzyme converts L-tryptophan into indole (the reverse reaction) and the resulting indole has toxic effects on the cells. The surfactant surrounds the indole to form a micelle, thereby rendering it non-toxic. Therefore, the surfactant is not added into the reaction mixture.
In another embodiment, the present invention relates to a method for preparing a halo-L-tryptophan wherein L-tryptophan or a surfactant are also present in the culture medium.
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.
DETAILED DESCRIPTION OF THE INVENTION
The microorganism employed in the present invention may be any microorganism capable of producing halo-L-tryptophan at a high optical purity from (a) a combination of haloindole, pyruvic acid and ammonia; or (b) a combination of haloindole and a source supplying pyruvic acid and ammonia. The source providing pyruvic acid and ammonia may be L-serine, L-cysteine, O-methyl-L-serine, O-benzyl-L-serine, S-methylcysteine, S-benzylcysteine, for example.
The microbial materials, regardless of origin or purity, may be employed as cells in the free state or as cells immobilized on a support such as by physical adsorption or entrapment. Immobilized cells or immobilized treated cell matter can be used, for example, by using entrapment in carrageenan or polyacrylamide or adsorption onto membranes of polyether sulfone or regenerated cellulose, for example. When intact cells are used in the present invention, the culture per se or intact bacterial cells collected from the culture may be used. The microbial cells may be used in the form of dried cells such as lyophilized, spray-dried or heat-dried cells, or in the form of treated cell matter.
As used herein, the term “treated cell matter” means the biological material, such as cell walls, membranes, nuclei, proteins, etc. which result from the subjection of intact cells to mechanical or chemical disruption. Preferred methods of disrupting intact cells by mechanical means include disruption by ultrasonication, glass beads, pressing and freeze-drying. Pressing in a French Press is particularly preferred. Preferred methods of disrupting intact cells by chemical means include disruption by lytic enzymes, organic solvents and surfactants. Following mechanical or chemical disruption the cell fragments and other cellular debris are removed by centrifugation or membrane filtration.
Crude enzyme fractions or purified enzymes prepared from the disruption of intact cells can also be used in the method of the invention, satisfactorily, when the enzyme fractions or purified enzymes retain the essential activity, and include crude enzyme fractions or purified enzymes prepared from treated cell matter.
Exemplary microorganisms for use in the invention include those belonging to the following genera which may be isolated from natural origins, such as plant or soil material: Proteus, Providencia and Morganella. Particularly preferred microorganisms are those species within the genus Proteus, such as
Proteus vulgaris
ATCC 13315
, Proteus mirabilis
ATCC 29906
, Proteus myxofaciens
, ATCC 19692 and
Proteus penneri
ATCC 33519. Particularly preferred microorganisms are also those species within the genus Providencia, such as
Providencia stuartii
ATCC 33672. Particularly preferred microorganisms are those species within the genus Morganella, such as
Morganella morganii
ATCC 8019.
It should be understood that mutants of the biologically pure microorganisms are also contemplated by the present invention for use in the methods described herein, such as those modified by the use of chemical, physical (for example, x-rays) or biological means (for example, molecular biology techniques).
Growth of the microorganisms may be achieved by one of ordinary skill in the art without undue experimentation by the use of an appropriate medium, including solid and liquid media. Methods for culturing the microorganisms in accordance with the invention can be facilitated in conventionally used culture media, namely culture media containing a carbon source, nitrogen source, inorganic salts, trace metal salts, and vitamins. Furthermore, depending on the species or culture conditions of the microorganism, the ability to produce halo-L-tryptophan can be promoted by adding L-tryptophan at a concentration of about 1.0 to 10.0 g/l to the cu
Onishi Norimasa
Yokozeki Kenzo
Ajinomoto Co. Inc.
Lilling Herbert J.
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