Heterologna ekspresija rekombinantnih enzima za razgradnju atrazina na površini stanične stijenke kvasca Saccharomyces cerevisiae

undergraduate thesis

• Title (english): Heterologous expression of the atrazine degradation pathway enzymes on the surface of yeast Saccharomyces cerevisiae cell
• Author: Fran Debanić
• Mentor: Renata Teparić (mentor)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Chemistry and Biochemistry); (Laboratory for Biochemistry); Zagreb
• Defense date and country: 2018-09-19, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Kvasac Saccharomyces cerevisiae jedan je od najčešće korištenih mikroorganizama u biotehnologiji, biomedicini i raznim znanstvenim istraživanjima. U ovom radu kvasac se koristio za ekspresiju enzima za razgradnju atrazina koji potječu iz Pseudomonas sp. ADP. Atrazin je herbicid koji je zabranjen od strane EU jer zbog svog kumulativnog efekta izaziva onečišćenje okoliša. Metabolički put razgradnje atrazina se sastoji od šest enzimskih reakcija od kojih je svaka katalizirana s po jednim enzimom. Cilj rada je bio eksprimirati i imobilizirati na površini stanične stijenke kvasca S. cerevisiae prva četiri enzima metaboličkog puta razgradnje atrazina (AtzA, AtzB, AtzC i AtzD), a u tu svrhu korišteni su proteini stanične stijenke kvasca Pir4 i Ccw12 s kojima su ti enzimi bili genetički fuzionirani. Uspješnost ekspresije i imobilizacije rekombinantih proteina provjeravala se Western blot analizom proteina ekstrahiranih iz izoliranih staničnih stijenki kvasca. Osim toga ispitivala se i aktivnost eksprimiranih rekombinantih enzima u tekućem mediju i na krutoj podlozi u Petrijevim zdjelicama.

Abstract (english)

Yeast Saccharomyces cerevisiae is one of the most commonly used microorganisms in biotechnology, biomedicine and various scientific research. In this thesis yeast was used to express the atrazine degradation enzymes originated from Pseudomonas sp. ADP. Atrazine is a herbicide that is banned from the EU because of its cumulative effect on environmental pollution. The metabolism of atrazine degradation consists of six enzymatic reactions each of which is catalysed with one enzyme. The aim of this study was to express and immobilize the first four enzymes of the atrazine degradation pathway (AtzA, AtzB, AtzC and AtzD) on the surface of the S. cerevisiae cell. For this purpose, Pir4 and Ccw12 yeast cell wall proteins were genetically fused with these enzymes. Success of expression and immobilization of the recombinant proteins was verified by Western blot analysis of proteins extracted from isolated yeast cell walls. In addition, the activity of expressed recombinant enzymes in the liquid medium and on the solid substrate in Petri dishes was also investigated.

• Keywords: Atrazin; Ccw12; Pir4; rekombinantni protein; Saccharomyces cerevisiae
• Keywords (english): Atrazine; Ccw12; Pir4; Recombinant protein; Saccharomyces cerevisiae
• Language: croatian
• URN:NBN: urn:nbn:hr:159:028171
• Study programme: Title: Biotechnology; Study programme type: university; Study level: undergraduate; Academic / professional title: sveučilišni/a prvostupnik/prvostupnica (baccalaureus/baccalaurea) inženjer/inženjerka biotehnologije (sveučilišni/a prvostupnik/prvostupnica (baccalaureus/baccalaurea) inženjer/inženjerka biotehnologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2018-09-24 07:56:17