Directed evolution of a glutaryl acylase into an adipyl acylase.
Semi-synthetic cephalosporin antibiotics belong to the top 10 of most sold drugs, and are produced from 7-aminodesacetoxycephalosporanic acid (7-ADCA). Recently new routes have been developed which allow for the production of adipyl-7-ADCA by a novel fermentation process. To complete the biosynthesis of 7-ADCA a highly active adipyl acylase is needed for deacylation of the adipyl derivative. Such an adipyl acylase can be generated from known glutaryl acylases. The glutaryl acylase of Pseudomonas SY-77 was mutated in a first round by exploration mutagenesis. For selection the mutants were grown on an adipyl substrate. The residues that are important to the adipyl acylase activity were identified, and in a second round saturation mutagenesis of this selected stretch of residues yielded variants with a threefold increased catalytic efficiency. The effect of the mutations could be rationalized on hindsight by the 3D structure of the acylase. In conclusion, the substrate specificity of a dicarboxylic acid acylase was shifted towards adipyl-7-ADCA by a two-step directed evolution strategy. Although derivatives of the substrate were used for selection, mutants retained activity on the beta-lactam substrate. The strategy herein described may be generally applicable to all beta-lactam acylases.
Sio
Charles F
CF
Pharmaceutical Biology, University Centre for Pharmacy, Groningen, the Netherlands; DSM-Gist, Delft, The Netherlands.
Riemens
Anja M
AM
van der Laan
Jan-Metske
JM
Verhaert
Raymond M D
RM
Quax
Wim J
WJ
D000581:Amidohydrolases / Q000378:metabolism; D000595:Amino Acid Sequence; D002511:Cephalosporins / Q000378:metabolism*; D003001:Cloning, Molecular; D019020:Directed Molecular Evolution; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010399:Penicillin Amidase / Q000235:genetics*; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics; D016415:Sequence Alignment; D013379:Substrate Specificity
D002511:Cephalosporins; C424182:adipoyl-7-amino-3-deacetoxycephalosporanic acid; D000581:Amidohydrolases; D010399:Penicillin Amidase; C046586:glutarylamidocephalosporanic acid acylase; C019665:amidase