Effects of catalytic site mutations on active expression of phage fused penicillin acylase.
Penicillin G acylase (EC 3.5.1.11) is 86-kDa large heterodimeric protein comprising two peptide A 23-kDa and peptide B 62-kDa, produced by intein-mediated auto-splicing of a 92-kDa precursor. Since penicillin G acylase was potentially employed in the preparation of a wide range of semi-synthetic beta-lactam antibiotics from acyl side-chain precursors and beta-lactam nucleus, directed evolution of penicillin acylase using phage display technology for extending its novel specificity is an interesting topic both of industry and academic. We fused the penicillin acylase to fd phage coat protein III and used pIII secretion signal sequence instead of penicillin acylase, which coupled gene and enzyme on phage particle and will be useful for directed evolution of penicillin acylase. Western blotting and enzyme activity assay were performed to demonstrate penicillin acylase has been functionally displayed on phage surface. Owing to the intimate association of enzyme activity and precursor processing in penicillin acylase, alterations of protein residues to make a phage library should be careful not to lead to processing defects. By site-directed mutagenesis, we have then identified effect of Ser B1 and Asn B241 variants on post-translational maturation of phage fused penicillin acylase.
Shi
Yi-Feng
YF
Department of Biotechnology, School of Biological and Food Engineering, Dalian Polytechnic University, No. 1 Qinggongyuan, Ganjingzi District, Dalian 116034, Liaoning, China. yifengshi@yahoo.cn
Soumillion
Patrice
P
Ueda
Mitsuyoshi
M
D017104:Bacteriophage M13 / Q000235:genetics*; D001665:Binding Sites; D002384:Catalysis; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D024861:Genetic Enhancement / Q000379:methods*; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D010399:Penicillin Amidase / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D010399:Penicillin Amidase