Glikozilacija i pH stabilnost penicilin G acilaze iz providencia rettgeri proizvedene u Pichia pastoris

Abstract

Penicilin G acilaza (PAC) je jedan od najšire korišćenih enzima u industrijskoj sintezi polusintetskih antibiotika. U ovom radu dobijeni nivo ekspresije PAC gena iz Providencia rettgeri u ekspresionom sistemu Pichia pastoris iznosio je 2.7 U/ml. Rekombinantni enzim je prečišćen i određen je njegov glikozilacioni status. Nađeno je da osim što su obe subjedinice enzima (α i β) N-glikozilovane, β subjedinica sadrži još i O-glikane. Takođe je ustanovljeno da je rekombinantna PACP. rett. stabilna u širokom pH opsegu što ju je, zajedno sa predhodno ustanovljenom visokom termostabilnošću, učinilo izuzetno privlačnim biokatalizatorom sa industrijske tačke gledišta.

Penicillin G acylase (PAC) is one of the most widely used enzymes in industrial synthesis of semi-synthetic antibiotics. The Providencia rettgeri pac gene was expressed to a level of 2.7 U/ml using the Pichia pastoris expression system. The recombinant enzyme was purified and its glycosylation status was determined. It was found that both subunits (α and β) of the enzyme were N-glycosylated, while the β-subunit also contained O-glycans. It was also observed that rPACP.rett. was stable in a wide range of pH, which, in addition to the previously proved high thermostability, makes it an attractive biocatalyst from an industrial point of view.