ИСТИНА

Penicillin acylase (EC 3.5.1.11) is widely used in industry for production of semi-synthetic penicillins and cephalosporins, as well as in fine organic synthesis and resolution of chiral compounds. Enzyme belongs to the superfamily of N-terminal nucleofile hydrolases. Gene of penicillin acylase from Alcaligenes faecalis VKM B-1518 (AfPA) was cloned and expressed in E. coli cells. AfPA contains eight unique amino acid substitutions compared to known penicillin acylases from other Alcaligenes faecalis strains. pac-Gene encodes precusor polypeptide that includes signal sequence, α-subunit, spacer and β-subunit. Complex multistep post-translational modification underlies bottlenecks of enzyme maturation. Processing comprises translocation of the precursor to the periplasm with concomitant removal of the signal peptide and consecutive excision of spacer. Active heterodimer is formed as the result. Our aim was to create new genetic construction that would enable enzyme production in simpler way and thereby would solve two important problems – standardization of expression conditions for any mutant and speed-up of the very expression. Three-dimensional structure analysis showed that N-terminus of α-subunit and C terminus of β-subunit are located in close proximity that opens up the possibility to connect two termini by short linker peptide. We conducted computer modeling of possible linkers and chose two the most perspective versions. In several PCR reactions we constructed gene encoding single-chain permuted penicillin acylase from Alcaligenes faecalis and created expression system in E. coli. After two-step purification we obtained homogenous preparation of the enzyme. Investigation of catalytic properties revealed close similarity of the new enzyme and wild-type AfPA. This work was supported by Russian Foundation for Basic Research (grant 13 04 01907).