ИСТИНА

Penicillin acylase (PA; EC 3.5.1.11) belongs to the superfamily of N-terminal nucleophile hydrolases and catalyzes cleavage of the amide bond in penicillin G and cephalosporin G. PA is used in industry for the production of semisynthetic β-lactam antibiotics. The gene of PA was cloned from several bacteria. New gene of PA from Alcaligenes faecalis strain VKM B-1518 (AfPA) was cloned in our laboratory. The enzyme displays higher thermal stability and wider pH-optimum of activity in comparison with the well-characterized PA from E.coli. AfPA was successfully expressed in E.coli. The characteristic feature of the wild-type AfPA is its two-step posttranslational processing. Previously, it was revealed that mutations can strongly affect the final protein yield, as well as the expression performance. Possible way to simplify protein maturation after translation and thus standardize expression conditions for mutant forms is preparation of the permuted enzyme. Permuted AfPA represents a single-chain polypeptide that is synthesized directly in the cytoplasm of E.coli without processing. Permuted AfPA was expressed in E.coli and characterized.