Fuctional Display Of The 85.6kDa Penicillin G Acylase On The Surface Of Phage Fd

It is firstly reported that the 85.6kDa heterodimeric penicillin G acylase from Bacillus megaterium was processed and functional displayed on the surface of phage fd. Phagemid pSurfscript was used to clone full-lenghth gene with amber stop codon of penicillin G acylase (signal peptide-α subunit-interaal peptide-β subunit) from Bacillus megaterium.A polypeptide with sequence of QKVDSSGGGGS was designed to be a linker between c terminal of penicillin G acylase and N terminal of the coat protein. The Ribosome Binding Site(RBS sequence) of pSurfscript is also replaced by RBS sequence originating from Bacillus subtilis. It was demonstrated that constructed phagemid can still express penicillin G acylase. E.coli XL1-blue cells were tansformed by pSurfpga and phages were rescued by M13KO7 helper phage particles. Results showed that the heterodimeric enzyme was expressed as a fusion protein that matures to an active biocatalyst connected to the coat protein of phage fd. An average value of 2.5×10-12m-units of enzyme activity was measured per cfu of phage. The display of penicillin G acylase from Bacillus megaterium not only offers the possibility of applying this technology for the selection of penicillin acylases with new side-chain specificities, but also facilitates our screen of mutant library constructed by using DNA shuffling technique.