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- Title
New strategy of site-directed mutagenesis identifies new sites to improve Streptomyces clavuligerus deacetoxycephalosporin C synthase activity toward penicillin G.
- Authors
Ji, Junjie; Tian, Xiuyun; Fan, Keqiang; Yang, Keqian
- Abstract
Based on multiple sequence alignment of different deacetoxycephalosporin C synthase (DAOCSs) and the crystal structure of Streptomyces clavuligerus DAOCS, 2-oxoglutarate, and penicillin G triple complex, ten residues (Y184, V245, S261, C37, T42, V51, S59, A61, Q126, and T213) not directly involved in substrate recognition were selected as mutational targets. Twenty one mutants were generated and characterized, and five (Q126M, T213V, S261M, S261A, and Y184A) showed improved activity toward penicillin G, with 1.45- to 4.50-fold increment in the k/ K. Q126, T213, and S261 are identified for the first time, as sites with significant effect on enzyme activity.
- Subjects
MUTAGENESIS; STREPTOMYCES clavuligerus; CEPHALOSPORINS; PENICILLIN; GENETIC mutation
- Publication
Applied Microbiology & Biotechnology, 2012, Vol 93, Issue 6, p2395
- ISSN
0175-7598
- Publication type
Article
- DOI
10.1007/s00253-011-3566-y