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..

Immobilization of alginate-PAC on Sepabeads EC-HA support

Penicillin acylase (PAC) is an important industrial enzyme for the production of many β-lactam antibiotics. It is capable of catalyzing the hydrolysis of penicillin G (Pen G) to generate phenylacetic acid (PAA) and 6-aminopenicillanic acid (6-APA). In this paper, in order to prevent enzyme inactivation, an attempt of coupling enzyme modification and immobilization was presented. Chemical modification was promoted to introduce carbohydrate moiety into the PAC molecule, capable of being covalently linked to an amino support. This seems to provide a possibility to couple the enzyme without risking a reaction at the active site which might cause a loss of activity. PAC molecules were modified by cross-linking with polyaldehyde derivatives of alginate in order to add them new and useful functions. Immobilization of alginate-PAC on Sepabeads EC-HA was used as a model system in order to demonstrate the potential of this strategy. Optimal conditions for covalent immobilization of alginate-PAC from Escherichia coli on support Sepabeads EC-HA, were investigated. The immobilized enzyme was then characterized by evaluating the potential effects of immobilization on its thermal stability, temperature and pH profile in comparison with native non-modified PAC and modified non-immobilized PAC. The maximum amount of the alginate-PAC coupled on the dry support of 99 mg/g was satisfactory. Deactivation rate constants at 50 ºC for free PAC, alginate-PAC and alginate-PAC immobilized on Sepabeads EC-HA were 2,32; 50,65 and 1,68 h-1, respectively. Alginate-PAC and alginate-PAC immobilized on Sepabeads EC-HA had the same pH and temperature optimum as the native non-modified PAC

Small farm business analysis using the ahp model for efficient assessment of distribution channels

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. Distribution of agricultural products from the aspect of farms is a continuous issue. Efficient application of the distribution model should provide a farm owner with regular income, as well as with a competitive advantage. Assessment of distribution channels in the context of an agricultural producer is subject to multi-criteria decisions, including both qualitative and quantitative factors. The aim of this paper is to develop a model which enables a structured analysis and an efficient assessment of distribution channels. The methodology based on the Analytical Hierarchy Process (AHP) will be a useful tool in setting various criteria for deciding on distribution channels, as well as a tool for managing a multi-variant qualitative assessment of data included in decision-making processes. The survey results show that the most important distribution channel for agricultural products is the channel of sales via processing plants (cooling and drying facilities, silos and agricultural combines), compared to sales through advertising (which significantly increased its share with the COVID-19 pandemic), sales through sales channels or at farmers’ markets

Design and characterization of alcalase-chitosan conjugates as potential biocatalysts

In this study, alcalase (protease from Bacillus licheniformis) immobilization by adsorption, enzyme crosslinking and covalent enzyme binding to activated chitosan microbeads were examined. The biocatalysts highest activity was obtained by covalent immobilization of alcalase onto a solid support. The alcalase covalent immobilization onto different types of chitosan beads obtained by inverse emulsion technique and electrostatic extrusion was studied. Parameters examined under different conditions were beads diameter, enzyme loading, enzyme capacity yield, and biocatalyst activity. The highest activity and enzyme loading of 23.6 IU/mg protein and 340.2 mg/g, respectively, were achieved by the enzyme immobilized onto chitosan microbeads obtained by the electrostatic extrusion technique. FT-IR analysis was used to confirm formation of alcalase-chitosan conjugates. The activity of optimally produced alcalase-chitosan microbeads was then verified in the industrially feasible reaction systems of egg white and soy protein hydrolysis. The high degree of hydrolysis of 29.85 +/- 0.967% after 180 min and five successive reuses obtained under real conditions (50 A degrees C, pH 8) verified the covalently bound alcalase to chitosan beads a promising candidate for use in industrial egg white protein hydrolysis process