Vpliv sekrecije heterolognih encimov kvasovke Saccharomyces cerevisiae na njihovo aktivnost

Bešić, Emina (author), Čadež, Neža (mentor), Kranjc, Luka (comentor)

Kvasovka Saccharomyces cerevisiae je tradicionalni biotehnološki organizem, ki se v zadnjih treh desetletjih uporablja za izražanje heterolognih proteinov na industrijskem nivoju. Prednost uporabe omenjenega organizma je sposobnost izločanja proteinov v gojišče, rast na preprostih substratih in nezahtevni pogoji kultivacije. Cilj naloge je bil testirati različna signalna zaporedja za izločanje fitaze v gojišče. Skonstruirali smo plazmida s konstitutivnim promotorjem PTEF1 in inducibilnim promotorjem PGAL1. Plazmida smo transformirali v sev S. cerevisiae INVSc1, kultivirali v sintetičnem gojišču brez uracila in z glukozo kot virom ogljika (SC-URA + GLU) ter primerjali zunajcelično in znotrajcelično fitazno aktivnost transformant s tema promotorjema. Pripravili smo plazmide s konstitutivnim promotorjem PTEF1 in s signalnimi zaporedji štirih različnih genov (MFα1, PLB1, CRH1, AGA2). Izmerili smo zunajcelično in znotrajcelično fitazno aktivnost v kompleknsem gojišču YPD in v gojišču SC-URA + GLU pri 28 °C in 37 °C. Pokazali smo, da vrsta gojišča vpliva na zunajcelično fitazno aktivnost. Sprememba temperature kultivacije ni imela vpliva na zunajcelično fitazno aktivnost. Ugotovili smo, da ima signalno zaporedje gena MFα1 v gojišču YPD najvišjo zunajcelično fitazno aktivnost in signalno zaporedje gena AGA2 najnižjo zunajcelično fitazno aktivnost.

• Language: Slovenian
• Keywords: kvasovke, Saccharomyces cerevisiae, rekombinantni proteini, fitaza, plazmidi, signalno zaporedje
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Publisher: [E. Bešić]
• Year: 2018
• PID: 20.500.12556/RUL-104951
• UDC: 602.3:582.282.23:604.4:577.15
• COBISS.SI-ID: 4971896
• Publication date in RUL: 18.10.2018

BEŠIĆ, Emina, 2018, Vpliv sekrecije heterolognih encimov kvasovke Saccharomyces cerevisiae na njihovo aktivnost [online]. Master’s thesis. E. Bešić. [Accessed 26 April 2025]. Retrieved from: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=eng&id=104951

Secondary language

• Language: English
• Title: The impact of the secretion of heterologous enzymes in yeast Saccharomyces cerevisiae on their activity

Abstract

The yeast Saccharomyces cerevisiae is a traditional biotechnological organism, which has been used for the production of different heterologous proteins on the industrial scale for the last three decades. Advantages of using S. cerevisiae for production of heterologous proteins are secretion of proteins into cultivation medium, and growth on simple substrates and under undemanding cultivation conditions. The aim of the study was to test different signal sequences for secretion of recombinant phytase into the culture medium. We constructed plasmids with a constitutive promotor PTEF1 and inducible promoter PGAL1. The plasmids were transformed into S. cerevisiae INVSc1, cultivated in the synthetic medium without uracil and with glucose (SC-URA + GLU). We compared extracellular and intracellular phytase activity of clones with these promoters. We prepared plasmids with a constitutive promoter PTEF1 and with signal sequences of four different genes (MFα1, PLB1, CRH1, AGA2). We measured extracellular and intracellular phytase activity in YPD rich medium and in SC-URA + GLU medium at 28 °C and 37 °C. We showed that the type of medium affects the measured extracellular phytase activity. Increased cultivation temperature did not affect the extracellular phytase activity. We found that the signal sequence of the gene MFα1 in the YPD medium has the highest extracellular phytase activity and the signal sequence of the gene AGA2 has the lowest extracellular phytase activity.

• Keywords: yeasts, Saccharomyces cerevisiae, recombinant proteins, phytase, plasmids, signal sequences