Razvoj primjene dviju glikozid hidrolaza - ekspresija saharoza fosforilaze na površini S. cerevisiae i praćenje aktivnosti alfa-amilaze

master's thesis

• Title (english): Development of the application of two glycoside hydrolases - expression of sucrose phosphorylase on S. cerevisiae surface and monitoring of alpha-amylase activity
• Author: Stela Lilek
• Mentor: Anita Slavica (mentor)
• Committee member: Renata Teparić (predsjednik povjerenstva)
• Committee member: Blaženka Kos (član povjerenstva)
• Committee member: Jasna Mrvčić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Biochemical Engineering); (Laboratory for Biochemical Engineering, Industrial Microbiology and Malting and Brewing Technology); Zagreb
• Defense date and country: 2022-09-30, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

U ovome radu je istražena mogućnost primjene dviju glikozid hidrolaza - saharoza fosforilaze i -amilaze. Primjenom odabranih metoda molekularne biotehnologije gen iz bakterije Leuconostoc mesenteroides koji kodira za saharoza fosforilazu (LmSPaza), umetnut je u plazmid pRST423_Ccw12_G i načinjen je konstrukt pRS_Ccw12LmSPaza. Ovi postupci su provedeni uz korištenje bakterijskog soja Escherichia coli DH5α, čije su transformirane stanice rezistentne na ampicilin. Gen koji kodira za LmSPazu insertiran je u konstruktu pRS_Ccw12LmSPaza između dva dijela gena ScCCW12, čime je osigurana uspješna ekspresija ovoga proteina na vanjskoj površini stijenke kvasca S. cerevisiae. LiAc metodom uspješno su konstruktom pRS_Ccw12LmSPaza transformirana dva soja kvasca S. cerevisiae - S. cerevisiae BY4741 i S. cerevisiae SEY6210, a ekspresija LmSPaze potvrđena metodama SDS-PAGE i Western blot. Hidroliza škroba α-amilazom uspješno je popraćena skenirajućom elektronskom mikroskopijom i na ovaj način su potvrđeni rezultati prethodnih istraživanja u ovoj grupi.

Abstract (english)

The use of two glycoside hydrolases - sucrose phosphorylase and -amylase was investigated in this thesis. By applying selected methods of molecular biotechnology, the gene from the bacterium Leuconostoc mesenteroides, coding for sucrose phosphorylase (LmSPase) was successfully inserted into plasmid pRST423_Ccw12_G and the pRS_Ccw12LmSpase construct was made. These procedures were carried out using the bacterial strain Escherichia coli DH5α, whose transformed cells are resistant to ampicillin. The gene that codes for LmSPase were inserted into the pRS_Ccw12LmSPase construct between two parts of the ScCCW12 gene, which ensured the successful expression of this protein on the outer surface of the S. cerevisiae yeast wall. Using the LiAc method, two S. cerevisiae strains - S. cerevisiae BY4741 and S. cerevisiae SEY6210 were successfully transformed with the pRS_Ccw12LmSPase and LmSPase expression was confirmed by SDS-PAGE and Western blot. Hydrolysis of starch by α-amylase was successfully monitored by scanning electron microscopy and in this way, the results of previous research in this group were confirmed.

• Keywords: glikozid hidrolaze; saharoza fosforilaza; -amilaza; Saccharomyces cerevisiae
• Keywords (english): glycoside hydrolases; sucrose phosphorylase; -amylase; Saccharomyces cerevisiae
• Language: croatian
• URN:NBN: urn:nbn:hr:159:993605
• Study programme: Title: Bioprocess Engineering; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra inženjer/inženjerka bioprocesnog inženjerstva (magistar/magistra inženjer/inženjerka bioprocesnog inženjerstva)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2023-02-27 11:19:11