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PVP-tyrosinase bioconjugates
Thesis (PhD)--Stellenbosch University, 2021.
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| Format: | Thesis |
| Language: | English |
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Stellenbosch : Stellenbosch University
2021
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| _version_ | 1867614092441681920 |
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| access_status_str | Open Access |
| author | Smit, Chandré Micaela |
| author2 | Klumperman, Bert |
| author_browse | Klumperman, Bert Smit, Chandré Micaela |
| author_facet | Klumperman, Bert Smit, Chandré Micaela |
| author_sort | Smit, Chandré Micaela |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (PhD)--Stellenbosch University, 2021. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/124231 |
| institution | Stellenbosch University (South Africa) |
| language | English |
| last_indexed | 2026-06-10T12:46:32.674Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2021 |
| publishDateRange | 2021 |
| publishDateSort | 2021 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/124231 PVP-tyrosinase bioconjugates Smit, Chandré Micaela Klumperman, Bert Pfukwa, Rueben Stellenbosch University. Faculty of Science. Dept. of Chemistry and Polymer Science. Povidone Cultivated mushroom Polymerization Enzyme kinetics Biocatalysis UCTD Thesis (PhD)--Stellenbosch University, 2021. ENGLISH ABSTRACT: The work presented herein describes the covalent surface modification of mushroom (Agaricus bisporus) tyrosinase by process of bioconjugation. Poly(N-vinyl pyrrolidone) (PVP) was selected as the synthetic polymer of choice, as it is a water soluble, biocompatible, non-toxic and chemically stable polymer. PVP has been identified as a promising alternative to poly(ethylene glycol) (PEG) for protein conjugation; and thus, its suitability for tyrosinase modification is systematically assessed in this study. The main aim of this study was to improve the thermal stability of mushroom tyrosinase, whilst retaining maximal enzyme activity. This was done in the hopes of overcoming the inherent limitations of tyrosinase for commercial use. A “grafting-to” approach was utilized for polymer attachment, with the Ɛ-amino groups of the enzyme surface lysine residues acting as the targeted reactive sites. Two synthetic routes towards lysine attachment were followed; route 1 implemented Schiff base conjugation chemistry and made use of an O-ethyl xanthate RAFT to carry out the controlled polymerization of NVP. This polymer was later successfully modified to yield the desired ω-aldehyde functionality. Route 2 was geared towards activated ester conjugation chemistry and utilized a RAFT agent with an N-hydroxysuccinimide (NHS) moiety directly built in at the α chain-end. This polymer was later modified to remove the reactive thiocarbonyl thio moiety, yielding α-NHS PVP with an unsaturated chain-end. PVP concentration and chain length were selected as variables for tyrosinase modification. Concentration studies showed that a maximal ratio of 5:1 (end-functional polymer: ε-amino group) was most favourable for bioconjugation. Above this concentration, a tendency for enzyme dissociation into its separate subunits was observed. Successful bioconjugation with PVP of varying chain lengths was confirmed by SDS-PAGE. Degrees of successful surface lysine modification varied from 30 - 40%. Changes in conjugate tertiary structure were observed using tryptophan fluorescence spectroscopy. Depending on the bioconjugation route utilized, the structural effects varied from “compressive” to “expansive”. The bioconjugate activity results showed that when route 1 (ω-aldehyde PVP) is followed in the absence of a reducing agent, the reversible imine linkage allows for significant activity enhancements, post-conjugation. A maximal kcat of 62 s¯¹ was obtained by this strategy, where the unmodified control showed a kcat = 36 s¯¹. Conversely, in the presence of a reducing agent for the same route, bioconjugate activity is greatly reduced, yielding a maximal kcat = 1.4 s¯¹. Route 2 (α-NHS PVP) indicated that modest activity enhancement is possible using the stable, secondary amino linkage. In this case, kcat = 39 s¯¹ was obtained. Thermal stability studies showed that the surface attachment of PVP to tyrosinase increased the enzyme Tₘ by 7 °C. This work showed that α-NHS PVP may be considered a suitable candidate for tyrosinase conjugation. This polymer yielded the shortest coupling reaction times, with 40% lysine attachment; and shows evidence for modest activity enhancement. In addition to this, enzyme thermal stability of the enzyme was also improved. Thus, rendering tyrosinase more compatible for commercial application, either for cosmetic or pharmaceutical end-use. AFRIKAANSE OPSOMMING: Die navorsing wat hier aangebied word, beskryf die kovalente oppervlakmodifikasie van sampioen- (Agaricus bisporus-)tirosinase deur die proses van biokonjugasie. Poli(N- vinielpirrolidoon) (PVP) is as die voorkeur- sintetiese polimeer gekies omdat dit ’n wateroplosbare, bioversoenbare, nietoksiese en chemies stabiele polimeer is. PVP word as ’n belowende alternatief vir poli(etileenglikol) (PEG) vir die doel van proteïenkonjugasie bestempel, en dus onderneem hierdie studie ’n stelselmatige evaluering van die geskiktheid van PVP vir tirosinasemodifikasie. Die hoofdoel van die studie is om die termiese stabiliteit van sampioentirosinase te verbeter, en terselfdertyd optimale ensiemaktiwiteit te behou. Dít word gedoen om die inherente beperkings van tirosinase vir kommersiële gebruik te prober oorkom. “Aanenting” (grafting to) is vir polimeerhegting gebruik en die Ɛ-aminogroepe in die oppervlaklisienreste van die ensiem het as die reaktiewe teikensetels gedien. Twee sintetiese roetes vir lisienhegting is gevolg: Roete 1 het konjugasiechemie met Schiff-basis geïmplementeer en van ’n O-etiel-xantaat-RAFT gebruik gemaak om die gekontroleerde polimerisasie van NVP uit te voer. Hierdie polimeer is later suksesvol gewysig om die gewenste ω-aldehied-funksionaliteit te verkry. Roete 2 het op konjugasiechemie met geaktiveerde ester berus, en het gebruik gemaak van ’n RAFT-agens met ’n direk ingeboude N-hidroksisuksienimied- (NHS-)helfte op die α-kettingpunt. Hierdie polimeer is later gewysig om die reaktiewe tiokarboniel-tio-helfte te verwyder, wat α-NHS-PVP met ’n onversadigde kettingpunt opgelewer het. PVP-konsentrasie en -kettinglengte is as veranderlikes vir tirosinasemodifikasie gekies. Konsentrasiestudies het op ’n maksimale verhouding van 5:1 (puntfunksionele polimeer : εaminogroep) as die gunstigste scenario vir biokonjugasie gedui. Bó hierdie konsentrasie is ’n geneigdheid tot ensiemdissosiasie en gevolglike aggregasie opgemerk. Suksesvolle biokonjugasie met PVP van wisselende kettinglengtes is deur middel van SDS-PAGE bevestig. Grade van suksesvolle oppervlaklisienmodifikasie het tussen 30% en 40% gewissel. Veranderinge in tersiêre konjugaatstruktuur is deur middel van triptofaanfluoressensiespektroskopie waargeneem. Die strukturele uitwerking was hetsy “saamdrukkend” of “uitsettend”, na gelang van die biokonjugasieroete wat gevolg is. Die resultate van die biokonjugaataktiwiteit het getoon dat wanneer roete 1 (ω-aldehiedPVP) sonder ’n reduseermiddel gevolg word, die omkeerbare imienkoppeling aansienlike aktiwiteitsverbetering ná konjugasie moontlik maak. ’n Maksimale kcat van 62 s-1 is met hierdie strategie verkry, terwyl die ongewysigde kontrole ’n kcat van 36 s-1 getoon het. Daarteenoor, wanneer dieselfde roete mét ’n reduseermiddel gevolg word, is biokonjugaataktiwiteit heelwat minder, met ’n maksimale kcat van 1.4 s-1. Roete 2 (α-NHSPVP) het aan die lig gebring dat matige aktiwiteitsverbetering moontlik is met behulp van die stabiele, sekondêre aminokoppeling. In dié geval is ’n kcat van 39 s-1 verkry. Termiese stabiliteitstudies het getoon dat die oppervlakhegting van PVP aan tirosinase die ensiemTm met 7 °C verhoog het. Hierdie navorsing toon dat α-NHS-PVP as ’n geskikte kandidaat vir tirosinasekonjugasie beskou kan word. Hierdie polimeer het die kortste koppelingsreaksietye getoon, met 40% lisienhegting, en lewer bewys van matige aktiwiteitsverbetering. Daarbenewens is die termiese stabiliteit van die ensiem verbeter, wat tirosinase dus meer geskik vir kommersiële toepassings maak. Doctoral 2021-07-12T08:55:17Z 2022-02-22T10:18:11Z 2021-07-12T08:55:17Z 2021-07 Thesis http://hdl.handle.net/10019.1/124231 en Stellenbosch University xx, 105 pages : illustrations application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Povidone Cultivated mushroom Polymerization Enzyme kinetics Biocatalysis UCTD Smit, Chandré Micaela PVP-tyrosinase bioconjugates |
| title | PVP-tyrosinase bioconjugates |
| title_full | PVP-tyrosinase bioconjugates |
| title_fullStr | PVP-tyrosinase bioconjugates |
| title_full_unstemmed | PVP-tyrosinase bioconjugates |
| title_short | PVP-tyrosinase bioconjugates |
| title_sort | pvp tyrosinase bioconjugates |
| topic | Povidone Cultivated mushroom Polymerization Enzyme kinetics Biocatalysis UCTD |
| url | http://hdl.handle.net/10019.1/124231 |
| work_keys_str_mv | AT smitchandremicaela pvptyrosinasebioconjugates |