Abstract
Penicillin acylase (PAC) is an important industrial enzyme for the production of many ?-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 ?C 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.
Highlights
Na osnovu dobijenih rezultata može se zaključiti da sintetski nosač Sepabeads EC-HA ima veliki kapacitet vezivanja za PAC, koja je prethodno modifikovana derivatom alginata, tako da se gotovo sva početna količina enzima vezuje za nosač (99 mg/g suvog nosača)
Optimal conditions for covalent immobilization of alginate-PAC from Escherichia coli on support Sepabeads EC-HA were investigated
Summary
Penicilin-acilaze iz E. coli (PAC) je dobijena od DSM (Holandija). Specifična aktivnost enzima je bila 105 U/cm, dok je sadržaj proteina određen metodom po Loriju (Lowry) iznosio 56,13 mg/cm proteina [19]. 1,07 g natrijum-perjodata (NaIO4) dodato je u 100 cm vodenog rastvora koji je sadržao 1 g alginata (50 mM krajnja koncentracija NaIO4) i reakciona smeša je inkubirana na 4 °C na tamnom mestu 48 h. Količina aktiviranog alginata u rastvoru konjugovanog enzima je određena fenol-sulfatnom metodom, kao što je opisano u literaturi [21]. Koncentracija proteina je određena metodom po Loriju korišćenjem goveđeg serum albumina kao standarda [19]. Koncentracija proteina i enzimska aktivnost u uzorcima je određena pre i posle imobilizacije. Aktivnost slobodne PAC, alginat–PAC i alginat–PAC–Sepabeads EC-HA je određena merenjem proizvoda enzimske hidrolize penicilina G, 6-APA, spektrofotometrijski sa PDAB kao što je prethodno opisano [23]. Jedna jedinica PAC je definisana kao količina enzima potrebna da se dobije 1 μmol/min 6-APA u uslovima analize (2% (w/v) rastvor penicilina G kao supstrata rastvorenog u 0,1 M fosfatnom puferu pH 7,92 na 37 °C). U predloženom kinetičkom modelu, promenljivi parametar je konstanta brzine dezaktivacije, kd, koja je određena nelinearnom Levenberg–Marquardt regresionom metodom korišćenjem Matlab softvera
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