TAILIEUCHUNG - Báo cáo khoa học: 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-aminodes-acetoxycephalosporanic 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-ADCAa highly active adipyl acylase is needed for deacylation of the adipyl derivative. Such an adipyl acylase can be generated from known glu-taryl acylases. | Eur. J. Biochem. 269 4495-4504 2002 FEBS 2002 doi Directed evolution of a glutaryl acylase into an adipyl acylase Charles F. Sio1 Anja M. Riemens2 Jan-Metske van der Laan2 Raymond . Verhaert1 i and Wim J. Quax1 1 Pharmaceutical Biology University Centre for Pharmacy Groningen the Netherlands 2DSM-Gist Delft the Netherlands Semi-synthetic cephalosporin antibiotics belong to the top 10 of most sold drugs and are produced from 7-aminodes-acetoxycephalosporanic 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 glu-taryl 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 b-lactam substrate. The strategy herein described may be generally applicable to all b-lactam acylases. Keywords 7-ADCA cephalosporin acylase directed evolution Pseudomonas SY-77 selection methods. Cephalosporin antibiotics belong to the most used drugs world-wide. The total global market for this class of b-lactams is included in the top 10 of most sold therapeutics surpassing the penicillin class of b-lactams 1 . Semisynthetic .

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