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master's thesis
Važnost aminokiselina Arg295 i Met367 za stabilnost enzima auksin-amidohidrolaze BrILL2 iz kupusa Brassica rapa ssp. pekinensis
Zagreb: University of Zagreb, Faculty of Science, 2018. urn:nbn:hr:217:708208
Rubić, Ivana
University of Zagreb
Faculty of Science
Department of Chemistry

Institutional repository: Repository of the Faculty of Science

Cite this document

Rubić, I. (2018). Važnost aminokiselina Arg295 i Met367 za stabilnost enzima auksin-amidohidrolaze BrILL2 iz kupusa Brassica rapa ssp. pekinensis (Master's thesis). Zagreb: University of Zagreb, Faculty of Science. Retrieved from https://urn.nsk.hr/urn:nbn:hr:217:708208

Rubić, Ivana. "Važnost aminokiselina Arg295 i Met367 za stabilnost enzima auksin-amidohidrolaze BrILL2 iz kupusa Brassica rapa ssp. pekinensis." Master's thesis, University of Zagreb, Faculty of Science, 2018. https://urn.nsk.hr/urn:nbn:hr:217:708208

Rubić, Ivana. "Važnost aminokiselina Arg295 i Met367 za stabilnost enzima auksin-amidohidrolaze BrILL2 iz kupusa Brassica rapa ssp. pekinensis." Master's thesis, University of Zagreb, Faculty of Science, 2018. https://urn.nsk.hr/urn:nbn:hr:217:708208

Rubić, I. (2018). 'Važnost aminokiselina Arg295 i Met367 za stabilnost enzima auksin-amidohidrolaze BrILL2 iz kupusa Brassica rapa ssp. pekinensis', Master's thesis, University of Zagreb, Faculty of Science, accessed 30 December 2024, https://urn.nsk.hr/urn:nbn:hr:217:708208

Rubić I. Važnost aminokiselina Arg295 i Met367 za stabilnost enzima auksin-amidohidrolaze BrILL2 iz kupusa Brassica rapa ssp. pekinensis [Master's thesis]. Zagreb: University of Zagreb, Faculty of Science; 2018 [cited 2024 December 30] Available at: https://urn.nsk.hr/urn:nbn:hr:217:708208

I. Rubić, "Važnost aminokiselina Arg295 i Met367 za stabilnost enzima auksin-amidohidrolaze BrILL2 iz kupusa Brassica rapa ssp. pekinensis", Master's thesis, University of Zagreb, Faculty of Science, Zagreb, 2018. Available at: https://urn.nsk.hr/urn:nbn:hr:217:708208

Metadata
TitleVažnost aminokiselina Arg295 i Met367 za stabilnost enzima auksin-amidohidrolaze BrILL2 iz kupusa Brassica rapa ssp. pekinensis
Title (english)The importance of Arg295 and Met365 amino acids for the stability of auxin-amidohydrolase BrILL2 enzyme from cabbage Brassica rapa ssp. pekinensis
AuthorIvana Rubić
MentorBranka Salopek Sondi (mentor)
Committee memberJasmina Rokov Plavec (predsjednik povjerenstva)
Committee memberVesna Petrović Peroković (član povjerenstva)
Committee memberBiserka Prugovečki (član povjerenstva)
Committee memberMarko Močibob (član povjerenstva)
GranterUniversity of Zagreb
Faculty of Science
(Department of Chemistry)
Zagreb
Defense date and country2018-06-18, Croatia
Scientific / art field,
discipline and subdiscipline		NATURAL SCIENCES
Chemistry
Abstract
Istražena je uloga aminokiselina R295 i M367 za stabilnost enzima auksin-amidohidrolaze BrILL2 iz kineskog kupusa (Brassica rapa L.). U tu svrhu korišteni su konstrukti R295A i M367A prethodno dobiveni mjesno-specifičnom mutagenezom. Rekombinantni proteini, divlji tip i mutirane varijante, su proizvedeni heterolognom ekspresijom u bakteriji Escherichia coli, pročišćeni afinitetnom kromatografijom pomoću Ni-NTA agaroze i provjereni elektroforezom u poliakrilamidnom gelu. Modeliranjem je predložen model, odnosno tercijarna struktura divljeg tipa BrILL2, a kao predložak korištena je auksin-amidohidrolaza iz Arabidopsis thaliana. Aminokiseline R295 i M367 stvaraju međusobnu interakciju u blizini aktivnog mjesta koja potpomaže postizanju stabilnije zatvorene konformacije enzima. Njihova mutacija u alanin dovodi do perturbacija u sekundarnoj strukturi mutiranih varijanata u odnosu na divlji tip što je dokazano metodom cirkularnog dikroizma. Diferencijalnom pretražnom kalorimetrijom pokazano je da promjene u sekundarnoj strukturi uzrokovane mutacijama utječu na stabilnost enzima.
Abstract (english)
The role of amino acids R295and M367was investigated for the stability of auxin-amidohydrolase BrILL2 from Chinese cabbage (Brassica rapa L.).Constructs R295A and M367A,previously prepared by site specific mutagenesis, were used. The recombinant proteins were produced by heterologous expression in Escherichia coli bacteria, purified by affinity chromatography using Ni-NTA agarose resin and confirmed by electrophoresis in polyacrylamide gel. Tertiary structure of wild type BrILL2 was modeled on the basis of auxin-amidohydrolase from Arabidopsis thaliana. Amino acids R295 and M367 interact in the proximity of the active site and this interaction participate in stabilization of closed conformation..Their mutation to alanine results in the perturbation of secondary structure of the mutants in comparison to the wild type which was proven by circular dichroism.Differential scanning calorimetry determined that the change of secondary structure affects the stability of the enzyme.
Keywords
Keywords (english)
Languagecroatian
URN:NBNurn:nbn:hr:217:708208
Study programmeTitle: Graduate university studies in chemistry; specializations in: Research programme
Course: Research programme
Study programme type: university
Study level: graduate
Academic / professional title: magistar/magistra kemije (magistar/magistra kemije)
Type of resourceText
File originBorn digital
Access conditionsOpen access
Terms of useLicence In copyright icon
Created on2018-06-27 09:32:42