Određivanje optimalnih uvjeta aktivnosti proteina YblCcw12p eksprimiranog u kvascu Saccharomyces cerevisiae

undergraduate thesis

• Title (english): Determination of optimal conditions for activity of protein YblCcw12p expressed in yeast Saccharomyces cerevisiae
• Author: Blanka Didak
• 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: 2011-06-29, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

U posljednje vrijeme raste interes za biotehnološku primjenu enzima imobiliziranih na površinu stanica domaćina koja tada ujedno djeluje kao producent i nosač imobiliziranog enzima. Od posebne važnosti su heterologni proteini vezani na staničnu stijenku kvasca Saccharomyces cerevisiae, budući da je kvasac nepatogeni mikroorganizam koji se lako transformira i uzgaja, a kao eukariot je pogodniji za ekspresiju enzima sisavaca nego bakterijske stanice. U ovom radu je karakterizirana enzimska aktivnost rekombinantne nukleaze YblCcw12p koja je dobijena fuzijom gena za intracelularnu nukleazu kvasca Yblp i gena za protein stanične stijenke Ccw12p preko kojeg je onda rekombinantni protein usidren u stijenku. U radu je pokazano da je rekombinantni enzim uspješno imobiliziran na površini stanice i da ima nukleaznu aktivnost. Za detekciju enzimske aktivnosti korištene su elektroforeza i SRED metoda koje su detaljno opisane u radu. Nadalje je provedena optimizacija metode za mjerenje enzimske aktivnosti modificiranjem metode po Kunitz-u, te su određeni optimalni uvijeti za aktivnost rekombinantnog enzima.

Abstract (english)

In the past decade interest is growing for the display of heterologous proteins on the surface of microorganisms, serving at the same time as producers and carriers of immobilized enzymes. Saccharomyces cerevisiae, as nonpathogenic microorganism which is easily transformed, cultivated, and better host for expression of mammalian enzymes than bacterial cells, has special importance in this field of biotechnology. In this work enzyme activity of recombinant nuclease YblCcw12p is detected and partially characterized. YblCcw12p is made by fusion of gene for yeast intracellular nuclease Yblp and gene for cell wall protein Ccw12p. Results presented in this work showed that recombinant enzyme is successfully immobilized on surface of the cell and posses nuclease activity. Electrophoresis and SRED method were used for detection of enzyme activity. Furthermore, enzyme activity was measured and optimal conditions for activity of recombinant protein were determined by modified and optimized Kunitz method.

• Keywords: rekombinantni protein; DNazna aktivnost; Saccharomyces cerevisiae
• Keywords (english): recombinant proteins; DNase activity; Saccharomyces cerevisiae
• Language: croatian
• URN:NBN: urn:nbn:hr:159:452223
• 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: Access restricted to students and staff of home institution
• Created on: 2017-05-19 08:00:23