Methionine Metabolism and Cephalosporin C Synthesis in Cephalosporium acremonium d-Amino Acid Oxidase

Aspergillus sp. strain O20 produces inducible D-amino acid oxidase intracellularly, only in the presence of some amino acids. The enzyme was induced most effectively by the addition of DL-alanine (1% w/v) to the production medium. Among the various compounds studied, production of the D-amino acid oxidase was enhanced by Aerosol-22, glucose, and sodium nitrate. D-Amino acid oxidase formation was observed during the onset of the stationary growth phase. Maximum enzyme activity was recorded after 96 h of fermentation (1000 IU/L).Key words: D-amino acid oxidase, Aspergillus sp., 7-aminocephalosporanic acid, cephalosporin C.

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Multipoint TvDAAO Mutants for Cephalosporin C Bioconversion

International Journal of Molecular Sciences ◽

10.3390/ijms20184412 ◽

2019 ◽

Vol 20 (18) ◽

pp. 4412

Author(s):

Denis L. Atroshenko ◽

Mikhail D. Shelomov ◽

Sophia A. Zarubina ◽

Nikita Y. Negru ◽

Igor V. Golubev ◽

...

Keyword(s):

Amino Acid ◽

Thermal Denaturation ◽

Catalytic Properties ◽

Acid Oxidase ◽

Cephalosporin C ◽

Wild Type ◽

Wild Type Enzyme ◽

Amino Acid Oxidase ◽

Catalytic Constant ◽

Biotechnological Processes

d-amino acid oxidase (DAAO, EC 1.4.3.3) is used in many biotechnological processes. The main industrial application of DAAO is biocatalytic production of 7-aminocephalosporanic acid from cephalosporin C with a two enzymes system. DAAO from the yeast Trigonopsis variabilis (TvDAAO) shows the best catalytic parameters with cephalosporin C among all known DAAOs. We prepared and characterized multipoint TvDAAO mutants to improve their activity towards cephalosporin C and increase stability. All TvDAAO mutants showed better properties in comparison with the wild-type enzyme. The best mutant was TvDAAO with amino acid changes E32R/F33D/F54S/C108F/M156L/C298N. Compared to wild-type TvDAAO, the mutant enzyme exhibits a 4 times higher catalytic constant for cephalosporin C oxidation and 8- and 20-fold better stability against hydrogen peroxide inactivation and thermal denaturation, respectively. This makes this mutant promising for use in biotechnology. The paper also presents the comparison of TvDAAO catalytic properties with cephalosporin C reported by others.

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Incorporation of 3H from δ-(l-α-amino (4,5-3H)adipyl)-l-cysteinyl-d-(4,4-3H)valine into isopenicillin N

Biochemical Journal ◽

10.1042/bj1840421 ◽

1979 ◽

Vol 184 (2) ◽

pp. 421-426 ◽

Cited By ~ 44

Author(s):

J O'Sullivan ◽

R C Bleaney ◽

J A Huddleston ◽

E P Abraham

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Adipic Acid ◽

Experimental Error ◽

Acid Oxidase ◽

Cephalosporium Acremonium ◽

Amino Acid Oxidase ◽

Free System ◽

A Cell ◽

Isopenicillin N

1. delta-(L-alpha-Amino[4,5-3H]adipyl)-L-cysteinyl-D-[4,4-3H]valine has been synthesized from its constituent amino acids, the L-alpha-amino[4,5-3H]adipic acid being obtained by reduction with 3H2 of methyl 5-acetamido-5,5-diethoxycarbonylpent-2-enoate and subsequent decarboxylation and hydrolysis. 2. In a cell-free system prepared by lysis of protoplasts of Cephalosporium acremonium 3H was incorporated from the doubly labelled tripeptide into a compound that behaved like penicillin N or isopenicillin N. The relative specific radioactivities of the alpha-aminoadipyl and penicillamine moieties of the penicillin were the same (within experimental error) as those of the alpha-aminoadipic acid and valine residues respectively of the tripeptide. 3. The behaviour of the labelled alpha-aminoadipic acid from the penicillin to the L-amino acid oxidase of Crotalus adamanteus venom showed that it was mainly L-alpha-aminoadipic acid. 4. The results are consistent with the hypothesis that the carbon skeleton of the LLD-tripeptide is incorporated intact into the penicillin molecule and that the first product is isopenicillin N.

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