prikaz prve stranice dokumenta Pozitivan učinak C-terminalnog rigidnog linkera na imobilizaciju saharoze fosforilaze
  Download
PDF 2.2 MB
master's thesis
Pozitivan učinak C-terminalnog rigidnog linkera na imobilizaciju saharoze fosforilaze
Zagreb: University of Zagreb, Faculty of Food Technology and Biotechnology, 2022. urn:nbn:hr:159:812823
Mikac, Ivan
University of Zagreb
Faculty of Food Technology and Biotechnology
Department of Biochemical Engineering
Laboratory for Biochemical Engineering, Industrial Microbiology and Malting and Brewing Technology

Institutional repository: Repository of the Faculty of Food Technology and Biotechnology

Cite this document

Mikac, I. (2022). Pozitivan učinak C-terminalnog rigidnog linkera na imobilizaciju saharoze fosforilaze (Master's thesis). Zagreb: University of Zagreb, Faculty of Food Technology and Biotechnology. Retrieved from https://urn.nsk.hr/urn:nbn:hr:159:812823

Mikac, Ivan. "Pozitivan učinak C-terminalnog rigidnog linkera na imobilizaciju saharoze fosforilaze." Master's thesis, University of Zagreb, Faculty of Food Technology and Biotechnology, 2022. https://urn.nsk.hr/urn:nbn:hr:159:812823

Mikac, Ivan. "Pozitivan učinak C-terminalnog rigidnog linkera na imobilizaciju saharoze fosforilaze." Master's thesis, University of Zagreb, Faculty of Food Technology and Biotechnology, 2022. https://urn.nsk.hr/urn:nbn:hr:159:812823

Mikac, I. (2022). 'Pozitivan učinak C-terminalnog rigidnog linkera na imobilizaciju saharoze fosforilaze', Master's thesis, University of Zagreb, Faculty of Food Technology and Biotechnology, accessed 26 August 2024, https://urn.nsk.hr/urn:nbn:hr:159:812823

Mikac I. Pozitivan učinak C-terminalnog rigidnog linkera na imobilizaciju saharoze fosforilaze [Master's thesis]. Zagreb: University of Zagreb, Faculty of Food Technology and Biotechnology; 2022 [cited 2024 August 26] Available at: https://urn.nsk.hr/urn:nbn:hr:159:812823

I. Mikac, "Pozitivan učinak C-terminalnog rigidnog linkera na imobilizaciju saharoze fosforilaze", Master's thesis, University of Zagreb, Faculty of Food Technology and Biotechnology, Zagreb, 2022. Available at: https://urn.nsk.hr/urn:nbn:hr:159:812823

Metadata
TitlePozitivan učinak C-terminalnog rigidnog linkera na imobilizaciju saharoze fosforilaze
Title (english)Positive effect of C-terminal rigid linker on immobilization of a sucrose phosphorylase
AuthorIvan Mikac
MentorAnita Slavica (mentor)
Committee memberRenata Teparić (predsjednik povjerenstva)
Committee memberBlaženka Kos (član povjerenstva)
GranterUniversity 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 country2022-09-30, Croatia
Scientific / art field,
discipline and subdiscipline		BIOTECHNICAL SCIENCES
Biotechnology
Abstract
Gene coding for sucrose phosphorylase (SP) from Leuconostoc mesenteroides (Lm) was inserted into pET21b with N- (N, His6-LmSP) or C-terminal (C) His₆-tag (LmSP-His₆) and rigid linker (L) consisted of repetitive sequences of (EAAAK)n with n = 6 (~5 nm) and 14 (~10 nm) or n = 6 (~5 nm), 14 (~10 nm) and 19 (~15 nm), respectively. Fused genes and the plasmid were used for electroporation of E. coli (BL21)DE3 and constructs were expressed, isolated, and purified by affinity chromatography. Since inclusion bodies were observed with N-terminally fused linkers, only Cterminally His6-tagged constructs with higher specific activity were considered for immobilization experiments. The immobilization of the control and LmSP-CL₅nm on SEPABEADSTM EC-EP was successful without any unbound protein in the supernatant. Due to its size, the rigid linker of LmSPCL₅nm completely prevented Cu²⁺(SEPABEADSTM EC-EP) and His₆ (the construct) coupling. The control and three LmSP-CL constructs were readily bound to Ni-NTA Superflow and had a higher immobilization yield than the control, with the highest immobilization efficiency of LmSP-CL₁₅nm.
Abstract (croatian)
Gen koji kodira saharozu fosforilazu (SF) iz Leuconostoc mesenteroides (Lm) je insertiran u pET21b sa N- (N, His₆-LmSF) ili C-terminalnim (C) His₆-tag-om (LmSF-His6) i rigidnim linkerom (L), koji je načinjen od ponavljajućih sekvenci (EAAAK)n, gdje je, redom, n = 6 (~5 nm) i 14 (~10 nm) ili n = 6 (~5 nm), 14 (~10 nm) i 19 (~15 nm). Geni fuzirani u plazmid korišteni su za elektroporaciju E. coli (BL21)DE3 i ovi konstrukti su eksprimirani, izolirani i pročišćeni afinitetnom kromatografijom. Inkluzijska tijela su formirana kod linkera na N-kraju proteina, pa su samo konstrukti sa His₆-tag-om na C-kraju proteina sa višom specifičnom aktivnosti korišteni u eksperimentima imobilizacije. Imobilizacija kontrole i LmSP-CL₅nm na nosaču SEPABEADSTM EC-EP bila je uspješna, te nevezani proteini nisu detektirani u supernatantu. Zbog svoje veličine, rigidni linker u LmSP-CL₁₅nm potpuno je spriječio povezivanje Cu²⁺(SEPABEADSTM EC-EP) i His₆ (s konstrukta). Kontrola i tri konstrukta LmSP-CL direktno su se vezali na Ni-NTA Superflow i, u usporedbi s kontrolom, uspješnija je bila imobilizacija ovih konstrukata, a najveća imobilizacijska učinkovitost je procijenjena kod LmSP-CL₁₅nm.
Keywords
Keywords (english)
Languageenglish
URN:NBNurn:nbn:hr:159:812823
Study programmeTitle: Molecular Biotechnology
Study programme type: university
Study level: graduate
Academic / professional title: magistar/magistra inženjer/inženjerka molekularne biotehnologije (magistar/magistra inženjer/inženjerka molekularne biotehnologije)
Type of resourceText
File originBorn digital
Access conditionsOpen access
Terms of useLicence Attribution-NoDerivatives 4.0 International (CC BY-ND 4.0) icon
Created on2023-02-27 12:44:47