ИСТИНА

Penicillin acylase (PA; EC 3.5.1.11) catalyses the hydrolysis of penicillin G to phenylacetic acid and 6-aminopenicillanic acid. PA is widely used in pharmaceutical industry for production of semi-synthetic penicillins and cephalosporins ТВ in fine organic synthesis for preparation of chiral compounds. In the case of PA majority of publications devoted to the enzyme from Escherichia coli (EcPA). However, PA from other bacterium, Alcaligenes faecalis, is superior over EcPA in many characteristics. In our laboratory new unique gene of PA from A.faecalis VKM B 1518 (AfPA) was cloned and overexpressed in E.coli. PA belongs to the superfamily of the N-terminal nucleophile hydrolases and comprises catalytic serine at the N-terminus of the β-subunit. Gene pac encodes inactive precursor composed of signal peptide, α subunit, spacer and β subunit. Precursor undergoes to complicated multistep posttranslational modification to form active αβ-heterodimer. In our laboratory protein engineering experiments with PA are carried out. It was found that in many cases single amino acid changes affect on maturation of mutant propeptide of active PA. Actually in the case of majority muteins, preparation of active mutant enzyme required separate optimization of cultivation conditions (including growth medium) of recombinant strain. Multi-step maturation of PA could be avoided by creation of permuted enzyme gene which will encode single-chain polypeptide with catalytic Ser residue at N-terminus. That enables to simplify process of enzyme formation due to the absence of processing. The aim of our work was creation and characterization of permuted PA from A.faecalis (pAfPA). Analysis of AfPA three-dimentional structure indicates that N-terminus of α subunit and C-terminus of β subunit are situated in close proximity. That fact makes it possible to connect these termini by linker peptide. We carried out structure computer modeling of pAfPAs with different linkers and two best candidates were selected. Polymerase chain reaction was used to construct pAfPA gene. Then pAfPA was expressed in E.coli as soluble and active enzyme. Optimization of cultivation conditions resulted in 15-fold increase of pAfPA yield. As well as standard purification scheme for wild-type AfPA did not allow to obtain homogenous preparation of pAfPA, the new procedure of pAfPA purification was developed out. Investigation of the properties of pAfPA showed similar values of catalytic parameters compared with the wild-type AfPA, but revealed approximately 10-fold higher thermal stability of pAfPA. Thermal inactivation kinetics of wt-AfPA and pAfPA were studied in wide range of different conditions (temperature, pH, buffer type and ion strength). This work was supported by Russian Foundation for Basic Research (grant 13 04 01907).