Prekomjerna ekspresija i biokemijska karakterizacija proteina dipeptidil-peptidaze III iz termofilne bakterije Caldithrix abyssi

Sučec, Iva

prikaz prve stranice dokumenta Prekomjerna ekspresija i biokemijska karakterizacija proteina dipeptidil-peptidaze III iz termofilne bakterije Caldithrix abyssi

master's thesis

Prekomjerna ekspresija i biokemijska karakterizacija proteina dipeptidil-peptidaze III iz termofilne bakterije Caldithrix abyssi

Zagreb: University of Zagreb, Faculty of Science, 2018. urn:nbn:hr:217:951364

Sučec, Iva

University of Zagreb
Faculty of Science
Department of Chemistry

Cite this document

APA 6th Edition

Sučec, I. (2018). Prekomjerna ekspresija i biokemijska karakterizacija proteina dipeptidil-peptidaze III iz termofilne bakterije Caldithrix abyssi (Master's thesis). Zagreb: University of Zagreb, Faculty of Science. Retrieved from https://urn.nsk.hr/urn:nbn:hr:217:951364

MLA 8th Edition

Sučec, Iva. "Prekomjerna ekspresija i biokemijska karakterizacija proteina dipeptidil-peptidaze III iz termofilne bakterije Caldithrix abyssi." Master's thesis, University of Zagreb, Faculty of Science, 2018. https://urn.nsk.hr/urn:nbn:hr:217:951364

Chicago 17th Edition

Harvard

Sučec, I. (2018). 'Prekomjerna ekspresija i biokemijska karakterizacija proteina dipeptidil-peptidaze III iz termofilne bakterije Caldithrix abyssi', Master's thesis, University of Zagreb, Faculty of Science, accessed 27 December 2024, https://urn.nsk.hr/urn:nbn:hr:217:951364

Vancouver

Sučec I. Prekomjerna ekspresija i biokemijska karakterizacija proteina dipeptidil-peptidaze III iz termofilne bakterije Caldithrix abyssi [Master's thesis]. Zagreb: University of Zagreb, Faculty of Science; 2018 [cited 2024 December 27] Available at: https://urn.nsk.hr/urn:nbn:hr:217:951364

IEEE

I. Sučec, "Prekomjerna ekspresija i biokemijska karakterizacija proteina dipeptidil-peptidaze III iz termofilne bakterije Caldithrix abyssi", Master's thesis, University of Zagreb, Faculty of Science, Zagreb, 2018. Available at: https://urn.nsk.hr/urn:nbn:hr:217:951364

Cite this item: https://urn.nsk.hr/urn:nbn:hr:217:951364

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Title	Prekomjerna ekspresija i biokemijska karakterizacija proteina dipeptidil-peptidaze III iz termofilne bakterije Caldithrix abyssi
Title (english)	Overexpression and biochemical characterization of protein dipeptidyl-peptidase III from thermophilic bacteria Caldithrix abyssi
Author	Iva Sučec
Mentor	Mihaela Matovina (mentor)
Committee member	Jasmina Rokov Plavec (predsjednik povjerenstva)
Committee member	Branimir Bertoša (član povjerenstva)
Committee member	Nives Galić (član povjerenstva)
Committee member	Marko Močibob (član povjerenstva)
Granter	University of Zagreb Faculty of Science (Department of Chemistry) Zagreb
Defense date and country	2018-07-17, Croatia
Scientific / art field, discipline and subdiscipline	NATURAL SCIENCES Chemistry
Abstract	Dipeptidil-peptidaze III (DPPIII) su egzopeptidaze koje prema MEROPS bazi podataka pripadaju obitelji metalopeptidaza M49. Kako bi se okarakterizirala porodica DPPIII i kod termofilnih organizmima, za ovaj rad odabran je član porodice DPPIII iz anaerobne, termofilne bakterije Caldithrix abyssi koja se može naći u morskim hidrotermalnim izvorima. Primijećeno je da prvi motiv aktivnog mjesta proteina CaDPPIII sadrži pentapeptidni motiv 'HEISH' umjesto karakterističnog heksapeptidnog motiva 'HEXXGH', dok je drugi motiv potpuno očuvan. Provedena je heterologna ekspresija kodirajućeg slijeda proteina DPPIII s histidinskim i glutation-S-transferaznim privjeskom. Rekombinantni protein pročišćen je afinitetnom kromatografijom i gel-filtracijom. Proteinu CaDPPIII-His6 određena je izoelektrična točka (pI = 6,12), te je nativnom gel-elektroforezom i izoelektričnim fokusiranjem pokazano postojanje dva različita strukturna oblika. Ispitivanje sintetskih supstrata peptidaza, pokazalo je značajnu hidrolitičku aktivnost enzima prema Arg-Arg-2-naftilamidu (Arg2-2NA), Gly-Arg-2NA, Phe-Arg-2NA i Pro-Arg-2NA. Ioni metala nemaju značajan utjecaj na peptidaznu aktivnost prema supstratu Gly-Arg-2NA. Najveća peptidazna aktivnost pokazana je kod pH = 7,0 i kod temperatura od 55 ˚C do 65 ˚C. Potpunu inhibiciju enzimske aktivnosti uzrokovali su kelirajući agensi, dok je djelomična inhibicija pokazana sa sulfhidrilblokirajućim reagensima. Kinetička mjerenja pokazala su da CaDPPIII kao supstrat preferira Arg2-2NA kao i drugi okarakterizirani enzimi ove porodice. U usporedbi s humanim, kvaščevim i enzimom iz Bacteroides thetaiotaomicron, Km enzima CaDPPIII je znatno veći, dok je konstanta brzine reakcije prvog reda koja odgovara pretvorbi supstrata u produkt (kcat) usporediva s istom konstantom enzima iz B. thetaiotaomicron. Dobiveni rezultati potvrđuju da je protein CaDPPIII član porodice DPPIII.
Abstract (english)	Dipeptidyl-peptidases III (DPPsIII) are exopeptidases that are classified as M49 family of metallopeptidases in MEROPS database. To better understand DPPsIII from thermophilic organisms, a protein from anaerobic, thermophilic bacteria Caldithrix abyssi was chosen for this thesis. These bacteria can be found in Mid-Atlantic Ridge hydrothermal vents. Protein CaDPPIII has a modified pentapeptide 'HEISH' active site motif, instead of characteristic 'HEXXGH' while the second motif is conserved. The gene for CaDPPIII with His-tag and GST-tag was heterologously expressed. The recombinant protein was purified with affinity and size exclusion chromatography. Isoelectric point (pI =6,12) was determined and two structural forms were detected by native electrophoresis and isoelectric focusing. Among various synthetic peptidase substrates examined, CaDPPIII-His6 preferred Arg-Arg-2-naphthylamide (Arg2-2NA), Gly-Arg-2NA, Phe-Arg-2NA and Pro-Arg-2NA. No significant enhancement in peptidase activity was observed in the presence of metal ions. Its hydrolytic activity was highest at pH = 7,0, and at 55 ˚C to 65 ˚C. Chelating agents completely inhibited the enzyme, while the sulfhydryl reagents inhibited it partially. Kinetic measurements showed that preferred substrate is Arg2-2NA, a characteristic DPPIII substrate. Constant Km is substantially higher for this enzyme compared to human, yeast and bacterial enzyme. First-order rate constant (kcat) is comparable with the same constant of the bacterial enzyme. Obtained results confirm CaDPPIII as a member of DPPIII family.
Keywords
Keywords (english)
Language	croatian
URN:NBN	urn:nbn:hr:217:951364
Study programme	Title: 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 resource	Text
File origin	Born digital
Access conditions	Open access
Terms of use
Repository	Repository of the Faculty of Science
Created on	2018-09-04 10:31:26