Evolution of an acylase active on cephalosporin C.
Semisynthetic cephalosporins are synthesized from 7-amino cephalosporanic acid, which is produced by chemical deacylation or by a two-step enzymatic process of the natural antibiotic cephalosporin C. The known acylases take glutaryl-7-amino cephalosporanic acid as a primary substrate, and their specificity and activity are too low for cephalosporin C. Starting from a known glutaryl-7-amino cephalosporanic acid acylase as the protein scaffold, an acylase gene optimized for expression in Escherichia coli and for molecular biology manipulations was designed. Subsequently we used error-prone PCR mutagenesis, a molecular modeling approach combined with site-saturation mutagenesis, and site-directed mutagenesis to produce enzymes with a cephalosporin C/glutaryl-7-amino cephalosporanic acid catalytic efficiency that was increased up to 100-fold, and with a significant and higher maximal activity on cephalosporin C as compared to glutaryl-7-amino cephalosporanic acid (e.g., 3.8 vs. 2.7 U/mg protein, respectively, for the A215Y-H296S-H309S mutant). Our data in a bioreactor indicate an ~90% conversion of cephalosporin C to 7-amino-cephalosporanic acid in a single deacylation step. The evolved acylase variants we produced are enzymes with a new substrate specificity, not found in nature, and represent a hallmark for industrial production of 7-amino cephalosporanic acid.
Pollegioni
Loredano
L
Dipartimento di Biotecnologie e Scienze Molecolari, Università degli Studi dell'Insubria, Via J.H. Dunant, 3, 21100 Varese, Italy. loredano.pollegioni@uninsubria.it
Lorenzi
Simona
S
Rosini
Elena
E
Marcone
Giorgia Letizia
GL
Molla
Gianluca
G
Verga
Roberto
R
Cabri
Walter
W
Pilone
Mirella S
MS
D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D002511:Cephalosporins / Q000378:metabolism*; D019020:Directed Molecular Evolution; D015723:Gene Library; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009154:Mutation / Q000235:genetics; D020836:Protein Structure, Quaternary; D013379:Substrate Specificity
D002511:Cephalosporins; C025163:cephalosporin C; D000581:Amidohydrolases; C019665:amidase