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
1999-02-05
2001-08-28
Marx, Irene (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...
C435S108000, C435S252100, C435S253300
Reexamination Certificate
active
06280979
ABSTRACT:
These racemic amino acids are often produced by chemical hydrolysis of DL-5-substituted hydantoins which can be synthesized easily from aldehydes by reaction with potassium cyanide and ammonium carbonate followed by resolution to obtain D(-)-&agr;-amino acids. However, these chemical methods involve complicated steps which are energy intensive, highly polluting and requiring the use of resolving agents.
The species of microorganisms exhibiting hydantoinase activity are Pseudomonas striata IFO 12996
; Peudomonas putida
DSM 84;
Agrobacterium radiobacter
, NRRL1[29]
; Alkalophilic Bacillus
sp. 123-3
Agrobacterium rhizogenes
IFO 13259. These microorganisms show the potentiality to convert prochiral hydantoins giving chiral D(-)-N-carbamoylaminoacids. [Yamada, Hikeak; Takahashi, Satomi; Yoneta, Kogi Kanegafuchi Chemical Industry Co. Ltd. Jpn Kokai 78,44690 (1978); U.S. Pat. No. 4,094,741 (1978); CA 89, 74216J, (1919); Degen, Ludwig; Viglia, Aurelio; Fascetti, Eugenio; Perricone, Elene SNAP Progeti Spn; Ger. Offen, 2,631,048 (1977); U.S. Pat. No. 4,111,749 (1978) CA: 86: 167160q (1977); R. Olivieri, F. Fascctti, L. Angelini and L. Degen., Biotech. & Bioeng., 23, 2173-2183 (1981); K. Soda, H. Tanaka, N. Esaki, Amino acids edited H. Dellweg, Biotechnology Vol. 3, 496 (1983), published: Verlag Chemie, Weinhein; S. Takahashi, acid production, editor H. Yamada et al. Elsevier Publications, Kodansha Ltd. Tokyo, Vol. 24, p. 269 (1986); A. Morin, W. Hummel and M. R. Kula., Applied Microbiology & biotechnology. 25, 91-96 (1986); H, Yamada, S. Shimizu, H. Shimada, Y. Tani, S. Takahashi, T. Ohashi, Biochemie, 62, 395-399 (1980), CA: 93, 68652 (1980); T. Mamoru, H. Takashi, T. Hitoshi, T. Shinichiro, M. Nobuyoshi, Jpn. Kokai 61,212,292 (1986), CA: 106, 100853 (1987); Kanegafuchi Chemical Industry, Jpn. Kokai 81,01,910 (1981), CA: 95, 40842 (1981); J. Kamphuis, W. H. J. Boesten, Q. B. Broxterman, H. F. M. Hermes, J. A. M. Balkem, E. M. Plexjer & H. E. Shoemaker; New Developments in chemo-enzymatic production of amino acids in Advances in Biochemical Engineering/Bio-technol. Vol. 42, 134-186, edited Aflechter, Publisher Springe-Verlag Berlin, Heidelber, 1990 ].
In all hitherto known processes a medium for the production of hydantionase consisted of basal mineral salts like KH
2
PO
4
, K
2
HPO
4
, MgSO
4
, MnSO
4
supplemented with beef extract, Yeast extract or peptone.
The D(-)-N-carbamoylaminoacid can be converted to D(-)-&agr;-amino acid by either chemical method [D. G. Sandro, P. Antonio, R. Luciano, Eur. Pat. 2,88,795 (1988), CA: 110, 193397f (1989); S. Takahashi; T. Ohashi, Y. Kii, H. Kumagai and H. Yamade, J. Ferment. Technol., 57, 328-332 (1979); O. Takehisa; F. Hirowka, T. satomi, N. Kenji, Jpn. Pat. 78,10,441 (1978), CA: 90, 39270 (1979)] or by enzymatic methods using N-carbamoylaminoacid amidohydrolase [T. Mamoru, T. Shinichiro HG. Takashi, T. Hitoshi, Jpn. Kokai 61,177,992 (1986), CA: 106, 1704lu (1987); R. Olivieri, E. Fascetti, L. Angelini, L. Degen, Enzyme Microbial Technol., 1, 201-204 (1979), CA: 91, 170502 (1979)] from Agrobacterium radiobacter, Arthrobacterium and Pseudomonas sps.
The prior art processes for decarbamoylation involve the use of enzyme (N-carbamoylaminoacid amido hydrolase) which is not readily available, while the chemical process involve the reaction of sodium nitrite in the presence of mineral acid or cation resin, where the product concentration is very low i.e. 1.5 to 2.5%. Concentration of aqueous solution in order to isolate the product from the reaction mixture is required which is energy-intensive process.
As compared to known process, the inventors by their R&D work developed an easier process for D(-)phenylglycine via D(-)-N-carbamoylphenylglycine from DL-5-phenylhydantion using bacterial cells as the biocatalyst. The strain Pseudomonas sp NCIM 5070) (ATCC 55940) used in these studies could grow well in the medium derived from cheap carbon sources like molasses and was able to produce 3-4% D(-)-N-carbamoylphenylglycine from DL-5-phenylhydantion within 6 hrs and its decarbamoylation with sodium nitrite in presence of mineral acid of appropriate strength.
The strain used in the present invention, Pseudomonas sp., was deposited under the Budapest treaty with the American Type Culture Collection (ATCC), now located at 10801 University Boulevard, Manassas, Va., 20110-2209, on Mar. 11, 1997, under Accession No. ATTC 55940, as well as with the National Collection of Industrial Microorganisms (NCIM), National Chemical Laboratory (NCL), Pune 411008, India, under Accession No. NCIM 5070.
The yield of the product in the invented process is 85-90% in each step and process involves the use of basic chemicals such as DL-5-phenylhydantion, molasses, sodium nitrite, mineral acid (sulphuric acid, hydrochloric acid) and D(-)-N-carbamoylphenylglycine.
The main objective of the present invention is to develop a improved process for the production of D(-)-phenylglycine from DL-5-phenylhyantion using D-hydantoinase.
Accordingly, this invention provides an improved and easier process for the production of D(-)-phenylglycine via D(-)-N-carbamoylphenylglycine from DL-5-phenylhydantion.
Salient features of the invention viz., the preparation of hydantoinase enzyme by growing the strain NCIM5070(ATCC5590) in molasses and used for the conversion of DL-5-phenylhydantion to D(-)N-carbamoylphenylglycine and its decarbamoylation is carried out with sodium nitrite in the optimized strength of sulphuric acid, where the product concentration is 7 to 10%, avoiding the concentration process of aqueous solution in order to isolate the product from reaction mixture. The product is isolated according to the process of this invention by adjusting the pH of the reaction mixture with sodium hydroxide/ammonium hydroxide.
The Pseudomonas strains available with us were screened along with a few isolates for the production of D(-)-N-carbamoylphenylglycine from DL-5-phenylhydantoin. Though 10 cultures showed hydantoinase activity to obtain D(-)-N-carbamoylphenylglycine from 5-phenylhydantoin, one of them exhibited higher hydantoinase activity after growing in molasses medium. Therefore, further studies on optimization of D(-)-N-carbamoylphenylglycine production were carried out using this strain of Pseudomonas sp. (NCIM 5070) (ATCC 55940). The culture gave high yields of D(-)-N-carbamoylphenylglycine (isolated yield 89%) as a solution of 3-4% strength. The conversion achieved is 89-92%. The train was catalase positive with optimum growth temperature between 25-28°. G=C contains of the DNA is 66-67 moles % (buoyant density). The denitrification does not occur.
The Pseudomonas cultures were individually grown in nutrient broth medium and the cells after centrifugation were used for the production of D(-)-N-carbamoylphenylglycine from DL-5-phenylhydantoin. The cultures giving good conversion (>75%) within short period (4 hrs) were further screened for their growth on molasses medium. Only one culture Pseudomonas sp. NCIM
5070
(ATCC 55940) exhibited better hydantoinase activity after growth in molasses medium. The optimization studies carried out using this strain showed that a temperature between 25-35° C. & pH range between 8.0-9.5 were more suitable for D(-)-N-carbamoylphenylglycine. Thus, D(-)-N-carbamoylphenylglycine was obtained within 4-6 hrs using alkaline buffer systems in 10 litre fermenter with a product concentration of 3-4%, which is further chemically converted to D(-)-phenylglycine.
REFERENCES:
patent: 5026906 (1991-06-01), DiGioacchino
patent: 2615594 (1976-10-01), None
Bastawde Kulbhushan Balwant
Gaikwad Bhaskar Ganapatrao
Gokhale Digambar Vitthal
Jogdand Vithal Venkatrao
Joshi Ramesh Anna
Council of Scientific & Industrial Research
Dinesh Agarwal, P.C.
Marx Irene
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