Isolation and mapping of a mutant penicillin G acylase with altered substrate specificity from Escherichia coli

1995 ◽  
Vol 17 (1) ◽  
pp. 19-23 ◽  
Author(s):  
Helmut Niersbach ◽  
Walter Tischer ◽  
Marijke Weber ◽  
Frank Wedekind ◽  
Roland Plapp
Keyword(s):  
Escherichia Coli ◽  
Substrate Specificity ◽  
Penicillin G Acylase ◽  
Penicillin G

scholarly journals Family Shuffling of Expandase Genes To Enhance Substrate Specificity for Penicillin G

2004 ◽  
Vol 70 (10) ◽  
pp. 6257-6263 ◽  
Author(s):  
Jyh-Shing Hsu ◽  
Yunn-Bor Yang ◽  
Chan-Hui Deng ◽  
Chia-Li Wei ◽  
Shwu-Huey Liaw ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Substrate Specificity ◽  
Kinetic Parameters ◽  
Substrate Inhibition ◽  
Fold Increase ◽  
Streptomyces Clavuligerus ◽  
Penicillin G ◽  
Dna Shuffling ◽  
Homologous Genes ◽  
Family Shuffling

ABSTRACT 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/Km 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.

scholarly journals Expression and regulation of the penicillin G acylase gene from Proteus rettgeri cloned in Escherichia coli.

1986 ◽  
Vol 168 (1) ◽  
pp. 431-433 ◽  
Author(s):  
G O Daumy ◽  
J A Williams ◽  
A S McColl ◽  
T J Zuzel ◽  
D Danley
Keyword(s):  
Escherichia Coli ◽  
Penicillin G Acylase ◽  
Penicillin G
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