| The first part: the goal of the first part is to obtain mutated DAOCSs (deacetoxycephalosporin C synthase, DAOCS) with increased expandase activity toward penicillin G, which are of potential use in the industrial production of 7-ADCA, an important precursor in the synthesis of cephalosporin antibiotics. The complex structure of DAOCS with penicillin G was analyized via RasWin and Swiss PDB Viewer. The residues in the substrate binding pocket and their interactions to the substrate penicillin G were identified. Eleven residues were chosen and their favorable mutations were designed. We combined eight of the eleven sites to four mutant libraries and the other three sites were used site-directed mutagenesis. The activities of these mutants were determined by bioassay and HPLC analysis, and activity increased mutants Y184A, S261A, S261M, R72T73, V72T73, T72T73 were obtained.The second part: the bifunctional deacetoxy/deacetylcephalosporin C synthase from Acremonium chrysogenum (acDAOC/DACS) catalyzes both the ring-expansion of penicillin N to deacetoxycephalosporin C (DAOC) and the hydroxylation of the latter to deacetylcephalosporin C (DAC). The residue R308, locating at the C-terminus of acDAOCS, showed significant effect on the enzymtic activity by controling substrates entry and products release. Saturation mutagenesis of R308 was performed, and four mutants, R308L, R308I, R308T and R308V, showed significant improvement in their ability to convert penicillin analogs. We concluded that replacement of R308 with amino acids that contain short hydrophobic side chains could improve the activity of acDAOCS for penicillin G. It may be due to that these amino acids mutations could decrease the polarity of the substrate/product channel. In this study, the apperent kcat and Km of purified R308L and R308I were determined via HPLC analysis, which showed similar Km but significant increased kcat. As a result, the kcat/Km of R308L and R308I increased 2.59- and 3.75-fold, respectively.
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