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Title:Imobilizacija MenD encima na mezoporozno siliko
Authors:ID Kovačič, Jure (Author)
ID Pečar, Darja (Mentor) More about this mentor... New window
ID Sudar, Martina (Comentor)
Files:.pdf VS_Kovacic_Jure_2026.pdf (2,99 MB)
MD5: 6AF1426271A18CCD0AFAC436B6AEBE8E
 
Language:Slovenian
Work type:Bachelor thesis/paper
Typology:2.11 - Undergraduate Thesis
Organization:FKKT - Faculty of Chemistry and Chemical Engineering
Abstract:V tej študiji smo se osredotočili na imobilizacijo 2-sukcinil-5-enolpiruvil-6-hidroksi-3-cikloheksen-1-karboksilata sintaze (MenD) na mezoporozno siliko SBA-15. MenD katalizira prvi odločilni korak pri biosintezi menakinona (vitamina K2) in zaradi svoje sposobnosti izvedbe encimsko katalizirane Stetterjeve reakcije predstavlja obetaven biokatalizator za organsko sintezo. Encim smo imobilizirali na mezoporozno siliko SBA-15, ki je bila izbrana kot nosilec zaradi svoje velike površine, dobro urejene strukture por in možnosti vgradnje funkcionalnih skupin na njeno površino. Te lastnosti spodbujajo učinkovito imobilizacijo encima, zagotavljajo boljši dostop substrata do aktivnega mesta in zmanjšujejo izpiranje encima iz nosilca. Sintetizirani mezoporozni nosilec SBA-15 smo karakterizirali s pomočjo vrstičnega elektronskega mikroskopa (SEM) za določitev morfologije delcev. Za določitev specifične površine, volumna por in porazdelitve velikosti por smo uporabili analizo po Brunauerju–Emmettu–Tellerju (BET), medtem ko smo za potrditev prisotnosti značilnih funkcionalnih skupin po funkcionalizaciji površine uporabili infrardečo spektroskopijo s Fourierovo transformacijo (FTIR). Učinkovitost imobilizacije smo ocenili z uporabo Bradfordovega testa za določanje beljakovin. Vzorce reakcijskega medija, reakcije s prostim ali imobiliziranim encimom, smo analizirali s tekočinsko kromatografijo visoke ločljivosti (HPLC). Merili smo porabo α-ketoglutarne kisline med reakcijo kot funkcijo časa. Iz rezultatov je razvidno, da je bila imobilizacija encima na nosilec delno uspešna. Ugotovili smo, da ima prost encim višjo katalitično učinkovitost v primerjavi z imobiliziranim encimom.
Keywords:encim, imobilizacija, mezoporozna silika, Stetterjeva reakcija
Place of publishing:Maribor
Year of publishing:2026
PID:20.500.12556/DKUM-99428 New window
Publication date in DKUM:14.09.2026
Views:274
Downloads:1
Metadata:XML DC-XML DC-RDF
Categories:KTFMB - FKKT
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Licences

License:CC BY 4.0, Creative Commons Attribution 4.0 International
Link:http://creativecommons.org/licenses/by/4.0/
Description:This is the standard Creative Commons license that gives others maximum freedom to do what they want with the work as long as they credit the author.
Licensing start date:17.08.2026

Secondary language

Language:English
Title:Immobilization of MenD enzyme on mesoporous silica
Abstract:In this study, we focused on the immobilization of 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate synthase (MenD) on mesoporous SBA-15 silica. MenD catalyzes the first committed step in the biosynthesis of menaquinone (vitamin K2) and, owing to its ability to catalyze the enzyme-mediated Stetter reaction, represents a promising biocatalyst for organic synthesis. The enzyme was immobilized on mesoporous silica SBA-15, which was selected as the support because of its high surface area, well-ordered pore structure, and the possibility of introducing functional groups onto its surface. These properties promote efficient enzyme immobilization, provide better substrate accessibility to the active site, and reduce enzyme leaching from the support. The synthesized SBA-15 mesoporous silica was characterized by scanning electron microscopy (SEM) to determine particle morphology. Brunauer–Emmett–Teller (BET) analysis was used to determine the specific surface area, pore volume, and pore size distribution, while Fourier-transform infrared (FTIR) spectroscopy was employed to confirm the presence of characteristic functional groups after surface functionalization. The immobilization efficiency was evaluated using the Bradford protein assay Samples of the reaction medium from reactions with free or immobilized enzyme were analyzed by high-performance liquid chromatography (HPLC). The consumption of α-ketoglutaric acid during the reaction was monitored as a function of time. The results showed that enzyme immobilization on the support was partially successful. The free enzyme exhibited higher catalytic efficiency compared with the immobilized enzyme.
Keywords:enzyme, immobilization, mesoporous silica, Stetter reaction


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