Effect of metal ions on activity of adenylosuccinate synthetase from bacterium Helicobacter pylori; The story of gelatin and fruit

Ovaj diplomski rad sastoji se od dva dijela: istraživačkog i metodičkog. U istraživačkom dijelu rada pročišćene su tri varijante enzima adenilosukcinat-sintetaze (AdSS) iz bakterije Helicobacter pylori: AdSS obilježen histidinskim privjeskom, AdSS kojem je aspartat 12 zamijenjen alaninom i AdSS kojem je aspartat 12 zamijenjen asparaginom. Za pročišćavanje su korištene kromatografija ionske izmjene, afinitetna kromatografija i gel filtracija. Proteinima su potom mjerene specifične aktivnosti, a za obiljeţen AdSS određeni su kinetički parametri uz promjenjivu koncentraciju iona Mg2+ koristeći Michaelis-Menten model enzimske kinetike. Konačno, ispitan je utjecaj različitih iona metala na aktivnost obilježenog AdSS. U metodiĉkom dijelu rada pod naslovom „Priča o želatini i voću“ izrađena je metodičkodidaktička priprema za učenike četvrtog razreda gimnazije. Metodička priprema temeljena je na obrazovnim ishodima, najčešćim učeničkim krivim shvaćanjima i na uvodu u relevantnu literaturu. Pripremljeni materijal namijenjen je obrazovnoj strategiji učenja otkrivanjem. This Diploma thesis consists of two parts: the research and methodological part. In the experimental part of diploma work, three variants of enzyme adenylosuccinate synthetase (AdSS) from bacterium Helicobacter pylori were purified: AdSS with histidine tag, AdSS with aspartate 12 changed to alanine and AdSS with aspartate 12 changed to asparagine. Ion exchange, affinity and gel filtration chromatographies were used for purification. Specific activity was measured for each enzyme variant, and for tagged AdSS kinetic parameters were also determined, by changing Mg2+ ions concentration, and using Michaelis-Menten model of enzyme kinetics. Finally, the effect of different metal ions on tagged AdSS activity was investigated. In the methodological part under the title “The story of gelatin and fruit” a didacticmethodological preparation was made for fourth grade high school students. The methodological preparation is based on the educational outcomes, common misconceptions held by students and with an insight in a revelant literature. The prepared material is intended for educational strategy of learning by discovery.