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Family Shuffling of Expandase Genes To Enhance Substrate Specificity for Penicillin G.
Jyh-Shing Hsu, 2004.Deacetoxycephalosporin C synthase (expandase) from Streptomyces clavuligerus, encoded by cefE, is an important industrial enzyme for the production of 7-aminodeacetoxycephalosporanic acid from penicillin G . To improve the substrate specificity for penicillin G, eight cefE-homologous genes were directly evolved by using the DNA shuffling technique . After the first round of shuffling and screening, using an Escherichia coli ESS bioassay, four chimeras with higher activity were subjected to a second round . Subsequently, 20 clones were found with significantly enhanced activity . The kinetic parameters of two isolates that lack substrate inhibition showed 8.5- and 118-fold increases in the k_cat/K_m ratio compared to the S . clavuligerus expandase . The evolved enzyme with the 118-fold increase is the most active obtained to date anywhere . Our shuffling results also indicate the remarkable plasticity of the expandase, suggesting that more-active chimeras might be achievable with further rounds .

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Last modified: May 25, 2005