ИСТИНА

Penicillin acylase (PA; EC 3.5.1.11) is an N-terminal nucleophile hydrolase that catalyzes cleavage of the amide bond in penicillins. The enzyme is used in industry for the production of semisynthetic β-lactam antibiotics. New penicillin acylase from Alcaligenes faecalis strain VKM B-1518 (wt-AfPA) was cloned in our laboratory. The gene of the wild-type AfPA encodes inactive precursor polypeptide that undergoes autoproteolytic processing to give active heterodimer. Cultivation experiments performed in our laboratory earlier revealed that introduction of point mutations can strongly affect the expression efficiency of mutant AfPAs. This work describes preparation and characterization of the permuted AfPA (pAfPA), alternative way for AfPA production. Circular permutation (CP) is a rearrangement of the polypeptide chain by connecting native N- and C-termini and creating new ends elsewhere. CP of proteins occurs in nature and is used in protein engineering for diverse purposes. The work includes computer modeling, construction of pAfPA gene, expression in E.coli, optimization of cultivation conditions, purification and comparative study of the properties.