Development of immobilized systems with penicillin acylase from Esherichia coli for production of semisyntetic penicillins

Osnovni cilj ove doktorske disertacije je bio optimizacija postupka dobijanja 6- aminopenicilanske kiseline primenom imobilisane penicilin-acilaze iz E. coli. Pri tome je razmatrana reakcija hidrolize prirodnog penicilina G do 6-APA katalizovane slobodnom i imobilisanom penicilin-acilazom. Da bi se realizovao postavljeni cilj bilo je potrebno izvršiti izbor nosača i metode za imobilizaciju enzima, optimizovati postupak imobilizacije enzima sa aspekta mase vezanog enzima i prinosa aktivnosti, okarakterisati dobijeni biokatalizator i ispitati razlike u kinetičkim parametrima slobodne i imobilisane penicilin-acilaze, izabrati odgovarajuće reaktorsko rešenje za izvođenje reakcije sa imobilisanim enzimima i ispitati operativnu stabilnost sistema. U prvom delu rada je izvršena karakterizacija slobodne penicilin-acilaze iz Escherichia coli i ispitana su njena katalitička svojstva u reakciji hidrolize penicilina G kao referentnom sistemu. Ova karakterizacija je bila neophodna da bi se utvrdile razlike u delovanju slobodnog i imobilisanog enzima. U tom cilju utvrđen je sadržaj proteina u komercijalnom enzimskom preparatu, specifična aktivnost, pH i temperaturni profil, termalna stabilnost, kao i vrednosti kinetičkih konstanti, i to Mihaelisove konstante i maksimalne brzine reakcije. Isto tako, ispitan je uticaj inhibicije supstratom u višku i proizvodima reakcije na brzinu reakcije u sistemu sa slobodnim enzimom i u tom cilju je određena vrsta inhibicije i vrednosti konstanti inhibicije. U drugom delu rada osnovni cilj istraživanja je bio usmeren na stabilizaciju enzima različitim postupcima. Pri tome je ispitano nekoliko postupaka hemijske imobilizacije enzima na različitim prirodnim i sintetskim polimerima (Sepabeads sa različitim funkcionalnim grupama i hitozan), kao i postupak imobilizacije prethodno hemijski modifikovanog enzima. U radu je ispitana mogućnost direktnog vezivanja penicilin-acilaze preko amino grupa u molekulu za epoksidne grupe nosača, zatim vezivanje enzima za nosač koji je prethodno aktiviran glutaraldehidom ili vezivanje prethodno modifikovanog enzima za nosače sa amino grupama...The aim of this work was the optimization of 6-aminopenicillanic acid obtaining procedure by using immobilized penicillin acylase from E. coli. The reaction of penicillin G hydrolysis to 6-APA catalyzed by free and immobilized penicillin acylase was considered. In order to realize the set aim, it was necessary to make a choice of carriers and methods for immobilization of the enzyme, to optimize enzyme immobilization procedure in terms of enzyme loading and activity yield. Also, the obtained biocatalysts were characterized and the differences in kinetic parameters of free and immobilized penicillin acylase were examined. An appropriate reactor solution for performing the reaction with the immobilized enzyme and the operational stability of the system were examined. In the first part of the thesis free penicillin acylase (PAC) from Escherichia coli was characterized and its catalytic properties were studied in the reaction of hydrolysis of penicillin G as a reference system. This characterization was necessary in order to determine the differences in the activities of the free and immobilized enzyme. Therefore, the protein content in the commercial enzyme preparation, specific activity, pH and temperature profile, thermal stability and the values of kinetic constants (Michaelis constant and maximal reaction rate) were determined. Likewise, the inhibition of PAC activity by substrate and reaction products (6-aminopenicillanic acid and phenylacetic acid) in the system with free enzyme was studied and types of inhibition and inhibition constant values were determined In the second part of the thesis the research has been focused on stabilizing the enzyme by different procedures. In with this aim, several procedures of chemical immobilization of the enzyme on various natural and synthetic polymers (Sepabeads with different functional groups and chitosan), as well as immobilization of the previously chemically modified enzyme were studied. In addition, possibilities of direct binding of penicillin acylase by amino groups in the enzyme to the epoxy groups of the carriers, the binding of the enzyme to the carriers activated with glutaraldehyde, as well as binding of the previously modified enzyme to the carriers with amino groups were investigated..