Izolacija in karakterizacija rekombinantnega encima paraoksonaze 1

Lavrič, Luka (author), Bavec, Aljoša (mentor)

Serumske paraoksonaze (PON) so proteinska družina pri sesalcih, ki jih sestavljajo encimi PON1, PON2 in PON3. Najbolj raziskan član družine je encim paraoksonaza 1 (PON1), ki ima laktonazno, arilesterazno in paraoksonazno aktivnost. Pomembno vpliva na preprečevanje srčno-žilnih in nevrodegenerativnih bolezni. V nalogi smo proizvedli rekombinanten PON1 (rePON1) v E. coli in ga izolirali z Ni2+-afinitetno in ionskoizmenjevalno kromatografijo. Sledila je detekcija z NaDS-PAGE in določitev koncentracije rePON1, ter določanje vplivov različnih substratov, metanola in inhibitorja 2-hidroksikinolina na stabilnost in aktivnost encima. Ugotovili smo, da metanol vpliva na stabilnost rePON1. Ob dodatku paraoksona ni bilo statistično pomembnega vpliva na stabilnost rePON1. Inhibitor 2-hidroksikinolin je inhibiral paraoksonazno aktivnost.

• Language: Slovenian
• Keywords: rekombinantna paraoksonaza 1, paraokson, 2-hidroksikinolin, stabilnost proteinov, encimska aktivnost
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2022
• PID: 20.500.12556/RUL-137577
• COBISS.SI-ID: 115519235
• Publication date in RUL: 22.06.2022

Secondary language

• Language: English
• Title: Isolation and characterization of recombinant paraoxonase 1

Abstract

Serum paraoxonases (PON) are family of enzymes PON1, PON2 and PON3. The most researched member of the family is the enzyme paraoxonase 1 (PON1), which has lactonase, arylesterase and paraoxonase activity. It has an important effect on the prevention of cardiovascular and neurodegenerative diseases. In this thesis, we produced recombinant PON1 (rePON1) in E. coli and isolated it with Ni2+-affinity and ion exchange chromatography. Then we detected rePON1 with SDS-PAGE and detected concentration of rePON1. We also measurement effects of substrates, methanol and inhibitor 2-hydroxyquinoline on enzyme stability and activity. We found that a percentage of methanol significantly affect on the stability of rePON1. With the addition of paraoxone there was no significant effect on the stability of rePON1. The 2-hydroxyquinoline inhibitor successfully inhibited paraoxonase activity.

• Keywords: recombinant paraoxonase 1, paraoxon, 2-hydroxyquinoline, protein stability, enzyme kinetics