Priprava rekombinantnih človeških citokromov P450 in njihovo biokatalizno vrednotenje v sintezi metabolitov izbranih učinkovin

Mihelčič, Kristina (author), Bratkovič, Tomaž (mentor), Glieder, Anton (co-mentor)

V okviru magistrskega dela smo optimizirali izražanje več človeških paralogov CYP450 v metilotrofni kvasovki Pichia pastoris. Med gojenjem kvasovk smo spreminjali stopnjo aeracije, pH gojišča in hitrost mešanja. Prav tako smo primerjali tudi rast celic med gojenjem v bioreaktorjih in erlenmajericah ter aktivnost encimov v suspenziji celic. Katalitično aktivnost CYP450 smo ovrednotili z inkubacijo modelnih učinkovin testosterona in diklofenaka v kulturah rekombinantnih kvasovk, ekstrakcijo metabolitov in njihovo analizo s HPLC.

• Language: Slovenian
• Keywords: Pichia pastoris, CYP 450, bioreaktorske enote, erlenmajerice, biotransformacija, HPLC, diklofenak, testosteron
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2020
• PID: 20.500.12556/RUL-124054
• Publication date in RUL: 23.12.2020

Secondary language

• Language: English
• Title: Production of recombinant human P450 cytochromes and their biocatalytic evaluation in selected drug metabolite synthesis

Abstract

As part of the master's thesis, we optimized the expression of several human CYP450 paralogs in the methylotrophic yeast Pichia pastoris. During the cultivation of yeasts, the degree of aeration, pH of the growth medium and mixing speed were modified. Also, yeast growth and enzyme activity following fermentation in bioreactors and shake flasks were compared. The catalytic activity of CYP450 was evaluated by incubation of the model drugs testosterone and diclofenac in recombinant yeast cultures, extraction of metabolites and their analysis by HPLC.

• Keywords: Pichia pastoris, CYP 450, bioreactor, shake flasks, biotransformation, HPLC, diclofenac, testosterone