O presente trabalho reporta a caracterização espectroscópica por absorção UV-visível, dicroísmo circular magnético (MCD) e ressonância paramagnética eletrônica (EPR) da proteína recombinante do tipoP450-canfora (P450CAM) codificada pela orf10 de Streptomyces clavuligerus (P450CLA) expressa por Escherichia coli Rosetta na forma nativa e associada a ligantes externos contendo os grupos β-lactamato, oxazol e derivado de alquilamina (álcool) do ácido clavulâmico. Considerando a diversidade de aplicações da enzima P450, sua reatividade com terc-butil-hidroperóxido (terc-BuOOH) foi caracterizada. O grupo heme da P450CLA apresenta-se covalentemente ligado e exibe características espectrais de UV-visível, CD e MCD de P450CAM, incluindo a banda Soret em 450 nm proveniente do complexo ferroso-CO. P450CLA foi convertido em espécies de valência alta pelo terc-BuOOH e promoveu cisão homolítica da ligação O-O. O perfil radicalar da reação foi terc-butiloxil como radical primário e metil e butilperoxil como radicais secundários. Os radicais metil e butilperoxil resultaram respectivamente da cisão-β do radical alcoxil e da reação do radical metil com oxigênio molecular.

The present study reports the spectroscopic characterization by UV-visible absorption spectroscopy, magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) of the recombinant orf10-encoded P450-camphor like protein (P450CLA)of Streptomyces clavuligerus expressed in Escherichia coli Rosetta in the native form and associated to external ligands containing the β-lactam, oxazole and alkylamine-derived (alcohol) moieties of the clavulamic acid. Considering the diversity of potential applications for the enzyme, the reactivity with tert-butylhydroperoxide (tert-BuOOH) was also characterized. P450CLA presents a covalently bound heme group and exhibited the UV-visible, CD and MCD spectral features of P450CAM including the fingerprint Soret band at 450 nm generated by the ferrous CO-complex. P450CLA was converted to high valence species by tert-BuOOH and promoted homolytic scission of the O-O bond. The radical profile of the reaction was tert-butyloxyl as primary and methyl and butylperoxyl as secondary radicals. The secondary methyl and butylperoxyl radicals resulted respectively from the β-scission of the alkoxyl radical and from the reaction of methyl radical with molecular oxygen.

The present article reviews different aspects of the chemistry of two widely used β-lactam antibiotics Clavulanic Acid and Cephamycin C. The article discusses important details of the biosynthesis of these compounds, their action mechanism and, principally, the methods employed in their isolation and purification, in accordance with the available literature. Despite the large quantity of available articles and patents concerning β-lactam antibiotics, those which describe the isolation and purification of Clavulanic Acid and Cephamycin C are rare. Overall, the intention of this article is to discuss the up-to-date scientific research related to the compounds under review.