Progress in One-pot Bioconversion of Cephalosporin C to 7-Aminocephalosporanic Acid.

BACKGROUND: Cephalosporins are the most widely used semisynthetic antibiotics, which acted on bacterial cell wall (peptidoglycan) synthesis. The key intermediate for fabricating about twothirds of cephalosporins in clinical use is 7-aminocephalosporanic acid (7-ACA), which is derived from chemical or enzymatic deacylation of the natural antibiotic cephalosporin C (CPC). The chemical deacylation process has been replaced by the enzymatic deacylation process because the chemical process required harsh conditions and released toxic waste. METHODS: A two-step enzymatic process that utilized D-amino acid oxidase (DAAO) and 7-ß-(4carboxybutanamido)-cephalosporanic acid acylase (GLA) for two successive reactions has been applied for the conversion of CPC to 7-ACA in an industrial scale. RESULTS: To simplify the process and lower costs, the one-pot enzymatic processes were developed by the application of the mono-enzymatic process (application of cephalosporin C acylase or the variants of GLA), di-enzymatic process (simultaneous action of DAAO and GLA) or the tri-enzymatic process (simultaneous action of DAAO, GLA and catalase) for direct conversion of CPC to 7-ACA. CONCLUSION: Here, we mainly focused on the description of these one-pot enzymatic processes and emphasized on the preparation of the involved biocatalysts.

Oxystress inducing antitumor therapeutics via tumor-targeted delivery of PEG-conjugated D-amino acid oxidase.

We had developed a H(2)O(2) generating enzyme, polyethylene glycol conjugated D-amino acid oxidase (PEG-DAO), which exhibited potent antitumor activity by generating toxic reactive oxygen species, namely oxidation therapy, subsequently showed remarkable antitumor effect on murine Sarcoma 180 solid tumor, by taking advantage of the enhanced permeability and retention effect. Along this line, we report here the preparation of PEG-DAO by use of recombinant DAO and its antitumor activity by using various tumor cell lines and tumor models. Recombinant DAO (rDAO) was obtained from E. coli BL21 (DE3) carrying the porcine DAO expression vector with high yield (20 mg/l) and high enzyme activity (5.3 U/mg). Pegylated rDAO (PEG-rDAO) showed high stability against sonication, repeated freezing/thawing, lyophilization and exhibited superior in vivo pharmacokinetics. PEG-rDAO had a molecular size of 65 kDa and existed as nanoparticles in aqueous solution with mean particle diameter of 119 nm. In vitro experiments showed strong cytotoxicity of PEG-rDAO against various tumor cells, whereas less cytotoxicity was found against various normal cells. In vivo antitumor treatment was carried out using 2 mice tumor models, namely colon 38 tumor and Meth A tumor model. PEG-rDAO was administered i.v. and after an adequate lag time, D-proline (the substrate of DAO) was injected i.p. to the tumor-bearing mice. Consequently, preferential generation of H(2)O(2) in the tumor was successfully achieved, which resulted in remarkable suppression of tumor growth without any visible side effects. These findings suggest a potential of PEG-rDAO as a novel anticancer strategy toward clinical development.

Synthesis, characterization and preliminary crystallographic data of N6-(6-carbamoylhexyl)-FAD-D-amino-acid oxidase from pig kidney, a semi-synthetic oxidase.

The FAD analogue, N6-(6-carboxyhexyl)-FAD, carrying a hexanoic acid residue at the N6 position of the adenine moiety was synthesized. A new semi-synthetic oxidase, N6-(6-carbamoylhexyl)-FAD-D-amino acid oxidase, was prepared by reacting the succinimido ester of N6-(6-carboxyhexyl)-FAD with apo-D-amino-acid oxidase from pig kidney in the presence of benzoate. Reaction conditions and methods have been developed for preparing pure semi-synthetic and fully active N6-(6-carbamoylhexyl)-FAD-D-amino acid oxidase that contains 1 covalently bound FAD analogue/subunit, as verified by redialysis, ultraviolet spectrophotometry, electrospray ionization (ESI)-MS and peptide mapping. Presumably, the N6-(6-carbamoylhexyl)-FAD moiety of this semi-synthetic D-amino-acid oxidase (DAAO), selectively bound to Lys163, has a structurally similar position to that of the non-covalently bound FAD of the native holoenzyme, since both DAAO forms show very similar kinetic properties (semi-synthetic DAAO, Vmax(app) = 17.7 mumol min-1 mg-1; KM(app) = 4.5 mM; native holo-DAAO, Vmax = 12.2 mumol min-1 mg-1; KM = 1.8 mM). Compared with the native holo-D-amino acid oxidase. this new semi-synthetic N6-(6-carbamoylhexyl)-FAD-D-amino acid oxidase is a considerably more stable enzyme that shows meso-thermostability and withstands inactivation on dilution. Probably, the lack of dissociation of FAD and, consequently, the absence of the instable apoenzyme are responsible for these phenomena. Preliminary investigations resulted in finding convenient and reproducible crystallization conditions for N6-(6-carbamoylhexyl)-FAD-D-amino acid oxidase. The single crystals, obtained by the sitting-drop method using ammonium sulfate as precipitant, belong to the tetragonal space group I422 with cell dimensions a = 16.3 nm, c = 13.6 nm. The crystals diffract to 0.3-nm resolution, with two molecules being present in the asymmetric unit, demonstrating the two-subunit quarternary structure of this semi-synthetic D-amino-acid oxidase.