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master's thesis
Pročišćavanje i djelomična karakterizacija rekombinantne ljudske dipeptidil-peptidaze III
Zagreb: University of Zagreb, Faculty of Food Technology and Biotechnology, 2010. urn:nbn:hr:159:201266
Andrejević, Marina
University of Zagreb
Faculty of Food Technology and Biotechnology
Department of Chemistry and Biochemistry
Laboratory for Biochemistry

Cite this document

Andrejević, M. (2010). Pročišćavanje i djelomična karakterizacija rekombinantne ljudske dipeptidil-peptidaze III (Master's thesis). Zagreb: University of Zagreb, Faculty of Food Technology and Biotechnology. Retrieved from https://urn.nsk.hr/urn:nbn:hr:159:201266

Andrejević, Marina. "Pročišćavanje i djelomična karakterizacija rekombinantne ljudske dipeptidil-peptidaze III." Master's thesis, University of Zagreb, Faculty of Food Technology and Biotechnology, 2010. https://urn.nsk.hr/urn:nbn:hr:159:201266

Andrejević, Marina. "Pročišćavanje i djelomična karakterizacija rekombinantne ljudske dipeptidil-peptidaze III." Master's thesis, University of Zagreb, Faculty of Food Technology and Biotechnology, 2010. https://urn.nsk.hr/urn:nbn:hr:159:201266

Andrejević, M. (2010). 'Pročišćavanje i djelomična karakterizacija rekombinantne ljudske dipeptidil-peptidaze III', Master's thesis, University of Zagreb, Faculty of Food Technology and Biotechnology, accessed 19 April 2024, https://urn.nsk.hr/urn:nbn:hr:159:201266

Andrejević M. Pročišćavanje i djelomična karakterizacija rekombinantne ljudske dipeptidil-peptidaze III [Master's thesis]. Zagreb: University of Zagreb, Faculty of Food Technology and Biotechnology; 2010 [cited 2024 April 19] Available at: https://urn.nsk.hr/urn:nbn:hr:159:201266

M. Andrejević, "Pročišćavanje i djelomična karakterizacija rekombinantne ljudske dipeptidil-peptidaze III", Master's thesis, University of Zagreb, Faculty of Food Technology and Biotechnology, Zagreb, 2010. Available at: https://urn.nsk.hr/urn:nbn:hr:159:201266

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Metadata
TitlePročišćavanje i djelomična karakterizacija rekombinantne ljudske dipeptidil-peptidaze III
Title (english)Purification and partial characterization of recombinant human dipeptidyl-peptidase III
AuthorMarina Andrejević
MentorVladimir Mrša (mentor)
GranterUniversity of Zagreb
Faculty of Food Technology and Biotechnology
(Department of Chemistry and Biochemistry)
(Laboratory for Biochemistry)
Zagreb
Defense date and country2010-06-07, Croatia
Scientific / art field,
discipline and subdiscipline		BIOTECHNICAL SCIENCES
Biotechnology
Abstract
Ljudska dipeptidil-peptidaza III (DPP III), član porodice metalopeptidaza M49, enzim je s ulogom u intracelularnom katabolizmu proteina. S ciljem istraživanja uloge evolucijski sačuvanog ostatka Asn406, u ovom diplomskom radu su sintetizirani divlji tip DPP III i njen mutirani oblik N406S prekomjernom ekspresijom u bakteriji Escherichia coli transformiranoj plazmidom koji je sadržavao cDNA za ljudsku DPP III s privjeskom od 6 His. Nakon pročišćavanja afinitetnom kromatografijom, rekombinantnim proteinima je određena molekulska masa (81 kDa) SDS-elektroforezom, specifična aktivnost za hidrolizu kromogenog supstrata Arg-Arg-2-naftilamida (37,81 jmg-1 za divlji tip, 14,52 jmg-1 za mutant N406S), optimalni pH (8,6) i temperatura djelovanja (37 °C), te temperaturna stabilnost (do 37 °C za divlji tip, do 30 °C za N406S). Ustanovljena je inhibicija obaju enzimskih oblika kelatorima metala i sulfhidrilnim reagensom p-hidroksimerkuribenzoatom, te selektivnost za supstrat Arg-Arg-2-naftilamid. Rezultati ukazuju na važnost Asn406 za postizanje pune katalitičke aktivnosti i za termičku stabilnost ljudske DPP III.
Abstract (english)
Human dipeptidyl-peptidase III (DPP III) is a member of the metallopeptidase family M49 with implied role in intracellular protein catabolism. To investigate the role of highly conserved Asn406, herein the wild-type DPP III and mutant N406S were overexpressed in Escherichia coli transformed with plasmid containing the cDNA encoding His6-tagged human DPP III. Recombinant proteins were purified by affinity chromatography, and their molecular mass (81 kDa) was determined by SDS-PAGE, as well as the specific activity for hydrolysis of chromogenic substrate Arg- Arg-2-naphthylamide (37.81 Umg-1, wild-type; 14.52 Umg-1, mutant N406S), pH and temperature optima (at 8.6, respectively 37 °C), and thermal stability (up to 37 °C for the wild-type, up to 30 °C for N406S). Both enzymes were inhibited by metallochelators and p-hydroxymercuri-benzoate, and showed selectivity for Arg-Arg-2-naphthylamide. The results suggest that Asn406 could be important for acquirement of full catalytic activity, and may contribute to human DPP III thermal stability.
Keywords
Keywords (english)
Languagecroatian
URN:NBNurn:nbn:hr:159:201266
Study programmeTitle: 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 resourceText
File originBorn digital
Access conditionsAccess restricted to students and staff of home institution
Terms of useLicence In copyright icon
RepositoryRepository of the Faculty of Food Technology and Biotechnology
Created on2017-08-29 09:32:48

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