Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase
Patent
1992-06-19
1993-05-04
Wax, Robert A.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Oxidoreductase
435814, 435816, 435179, C12N 906, C12N 1112
Patent
active
052081559
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a D-amino acid oxidase which is active against cephalosporin C, and a method for isolating it from Trigonopsis variabilis.
There is a long-standing considerable interest in using amino acid oxidase activity for forming o-keto acids from their corresponding amino acids since these are of interest as a nutritional supplement for patients suffering from kidney insufficiency. Both D-amino acid oxidase activity found in the yeast Trigonopsis variabilis (Brodelius, P., Nilsson, K., and Mosbach, K., 1981, Appl. Biochem, Biotechnol. 6, 293-308) and L-amino acid oxidase activity found in the bacterium Providencia (Szwajcer, E., Brodelius, P., and Mosbach, K. (1982). Enzyme Microb. Technol. 4, 409-413) have been used. D-amino acid oxidase also exhibited activity against cephalosporin C, by oxidatively deaminating the latter to keto adipic 7-aminocephalosporanic acid. The same activity was also reported in U.S. Pat. No. 3,658,649. However, the extract used was obtained only by ammonium sulfate precipitation and thus represents crude preparation comprising several amino acid oxidases A number of different organisms have been examined in respect of the latter activity, including E. coli, Pseudomonas species, Aerobacter species, Candida tropicalis, Penicillium rocforti, Aspergillus flavus and A. niger, Neurospora crassa, Nocardia, Citrobacter and Trigonopsis variabilis. Only Trigonopsis variabilis and Citrobacter could deaminate cephalosporin C to keto adipic 7-aminocephalosporanic acid. The activity found in Citrobacter was very low and appeared to be membrane-bound, while the enzyme from Trigonopsis was present in the cytoplasm and at a much higher level. It should be mentioned in this context that the activity against cephalosporin C was also found with the enzyme obtained from hog kidney (Mazzeo, P., and Romeo, A. (1972). J. C. S. Perkin I(P3), 2532). 7-Aminocephalosporanic acid has great industrial interest as basic moiety for the preparation of semisynthetic cephalosporins by analogy with 6-aminopenicillanoic acid for the preparation of semisynthetic penicillins. An acylase has been reported in the literature (Shibyva, Y., Matsumoto, K., and Fuji, T. (1981). Agr. Biol. Chem. 45, (1561-1567) which hydrolyzes the side-chain of glutaryl-7-aminocephalosporanic acid. The latter compound is spontaneously formed from keto adipic-7-aminocephalosporanic acid by the hydrogen peroxide simultaneously formed. The following diagram illustrates the formation of 7-aminocephalosporanic acid, under the influence of two enzymes: ##STR1##
The present invention has for its object to produce a Trigonopsis variabilis D-amino acid oxidase in substantially pure form and active against cephalosporin C.
Another object of the invention is to provide a method for isolating a D-amino acid oxidase from Trigonopsis variabilis.
The simple method for purifying D-amino acid oxidase to homogeneity according to the invention is carried out in three steps. Thus, the method is characterized in that it comprises to obtain a precipitate and supernatant fraction; ammonium sulfate to obtain a second precipitate, said second precipitate containing the D-amino acid oxidase of claim 1; and D-amino acid oxidase by isoelectric precipitation.
The acidification according to step (a) above can be performed by adding acetic acid to the crude cell extract. The cell extract is acidified to a pH of about 4 to 6, preferably about 5.1 to 5.3 and most preferably about 5.3.
In a further embodiment, any precipitate formed after acidification is removed prior to heating. Further, D,L-methionine can be added prior to heating, preferably in an amount to obtain a final concentration of about 25 mM.
The crude extract is heated in step (a) above to a temperature of about 40.degree. to about 60.degree. C., preferably about 40.degree. to about 50.degree. C. and most preferably to about 50.degree. C.
In another preferred embodiment, the supernatant fraction of step (a) is dialyzed prior to the treatment with ammonium sulfate, preferably against a bu
REFERENCES:
patent: 3658649 (1972-04-01), Arnold et al.
patent: 3801458 (1974-04-01), Fildes et al.
patent: 4486549 (1984-12-01), Matsumoto et al.
Berg, C. P., et al. (1976) Anal. Biochem, 71, 214-222.
Biotechnology Applications and Research (eds. Cheremisino P. N., et al., Technomil Pub. Co., Inc., 1985, pp. 21, 541-557 Szwajcer F., et al. (1985) Biotechnol. Lett. 7(1), 1-7.
Mosbach Klaus
Szwajcer Estera
Hendricks Keith D.
Wax Robert A.
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