Karakterizacija 5'-fosfodiesteraze iz sladnih klica

master's thesis

• Title (english): Characterization of purified 5´-phosphodiesterase from malt sprouts
• Author: Andrea Jelić
• Mentor: Sunčica Beluhan (mentor)
• 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: 2005-12-13, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

U ovom radu je provedena karakterizacija potpuno pročišćene 5´-fosfodiesteraze, nakon što je ekstrahirana iz sladnih klica u Tris-HCl puferu pH 8,9 te dalje pročišćena dijalizom, ionskom izmjenom i gel elektroforezom. Karakterizacija je provedena ispitivanjem utjecaja dvovalentnih kationa, EDTA i NaN3, temperature i pH vrijednosti na aktivnost enzma. Također je ispitivana supstratna specifičnost, kao i inhibicija produktima hidrolize, 5´-ribonukleotidima (5´-AMP, 5´-GMP, 5´-UMP, 5´-CMP i 5´-IMP). Potpuno pročišćeni enzim imao je pH optimum 8,9, te je pokazao maksimalnu aktivnost pri 70 °C. Također je najveću aktivnost pokazao dodatkom 10 mM Mg2+ iona, dok su mu Zn2+, Cu2+ i Mn2+ ioni, EDTA i NaN3 smanjili aktivnost. Izolirana 5´-fosfodiesteraza je pokazala Km vrijednost 0,28 mM za timidin 5´-monofosfat p-nitrofenil ester (supstrat za 5´-fosfodiesterazu). Nadalje, enzim je pokazao iznimnu sposobnost hidrolize RNA i jednolančane DNA, te vrlo slabi afinitet prema dvolančanoj DNA.

Abstract (english)

In this work 5´-phosphodiesterase from malt sprouts was extracted in Tris-HCl buffer pH 8,9, and then was purified by dialysis, ion exchange and SDS-PAGE, and further on characterized. Purified enzyme had an optimum pH of 8,9 and maximum activity at 70 °C. In the presence of 10 mM Mg2+ ions, increased activity was noted, while Zn2+, Cu2+ and Mn2+ ions, EDTA , NaN3 and 5´-ribonucleotides (5´-AMP, 5´-GMP, 5´-UMP, 5´-CMP and 5´-IMP) decreased the enzyme activity. It had a Km of 0,28 mM on the hydrolysis of thymidine 5´-monophosphate p-nitrophenyl. This enzyme was able to hydrolyze RNA and single stranded DNA, whereas double stranded DNA was not a good substrate.

• Keywords: 5´-fosfodiesteraza; sladne klice
• Keywords (english): 5´-phosphodiesterase; malt sprouts
• Language: croatian
• URN:NBN: urn:nbn:hr:159:781533
• Study programme: Title: Biotechnology; specializations in: Biochemical Engineering; Course: Biochemical Engineering; Study programme type: university; Study level: graduate; Academic / professional title: diplomirani inženjer biotehnologije; smjer biokemijsko inženjerstvo (diplomirani inženjer biotehnologije; smjer biokemijsko inženjerstvo)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Access restricted to students and staff of home institution
• Created on: 2017-09-11 13:31:18