In vitro conversion of penicillin G and ampicillin by recombinant Streptomyces clavuligerus NRRL 3585 deacetoxycephalosporin C synthase

The in vitro conversion of penicillin N analogues to cephalosporins was fir st demonstrated in resting cells and cell-free extracts of Streptomyces cla vuligerus NP1. This study has optimized the conversion rate of a highly exp ressed soluble source of recombinant S. clavuligerus NRRL 3585 deacetoxycep alosporin C synthase (scDAOCS) through detailed characterization of its cof actor requirements. Inclusion of dithiothreitol (DT-F) and ascorbate at var ious concentrations from 0.2-16 mM and 0.2-10 mM was found to be inhibitory to scDAOCS activity under specified reaction conditions examined. Simultan eous omission of these cofactors in a new modified reaction condition. comp rising of 50 mM Tris HCl (pH7.4), 0.8 mM ATP, 1.28 mM alpha -ketoglutarate and 1.8 mM FeSO4, enhanced the conversion rate of two penicillin N analogue s. penicillin G and ampicillin, through 20- and 35-fold respectively. (C) 2 001 Elsevier Science Inc. All rights reserved.