Priprava in uporaba kombiniranih zamreženih encimskih agregatov v mikropretočnih sistemih za biokatalitsko sintezo δ-viniferina

Tomažin, Natalija (author), Žnidaršič Plazl, Polona (mentor)

V magistrskem delu obravnavam pripravo in uporabo kombiniranih zamreženih encimskih agregatov (angl. crosslinked enzyme aggregates, CLEAs) v mikropretočnem sistemu za biokatalitsko sintezo δ-viniferina iz resveratrola. Študija temelji na povezovanju biokatalitske reakcije oksidacije resveratrola s H$_2$O$_2$, katalizirane s hrenovo peroksidazo (HRP), z in situ sintezo tega substrata na osnovi reakcije, katalizirane z glukoza oksidazo (GOx), kar zmanjšuje inhibitorni učinek H$_2$O$_2$ na HRP. Resveratrol je naravna fenolna spojina, prisotna v grozdju in s tem tudi v tropinah, ki nastajajo kot stranski produkt pri pridelavi vina. Tropine so pomemben agroindustrijski odpadek, saj vsebujejo biološko aktivne spojine. Njihova uporaba kot surovine predstavlja primer valorizacije odpadkov, saj omogoča pretvorbo nizkovrednega ostanka v produkt z dodano vrednostjo, kot je δ-viniferin. V okviru dela smo razvili mikropretočni sistem za pripravo zamreženih encimskih agregatov HRP (CLEA-HRP) ter kombiniranih zamreženih encimskih agregatov HRP in GOx (CLEA-HRP-GOx). Mikropretočni pristop omogoča natančen nadzor nad pogoji obarjanja in zamreževanja, kar vodi do bolj enotne velikosti delcev in izboljšane zadržane encimske aktivnosti v primerjavi s klasičnimi šaržnimi postopki. Optimizirali smo pogoje priprave, kot so zadrževalni čas, vrsta in koncentracija obarjalnega ter zamreževalnega sredstva ter izbira pufra. Encimski pripravek CLEA-HRP-GOx smo uporabili v šaržni biotransformaciji resveratrola. Potek reakcije smo spremljali z NMR spektroskopijo. Primerjali smo aktivnosti obeh encimov v zamreženih agregatih s prostimi encimi. Rezultati kažejo, da CLEA-HRP-GOx omogočajo učinkovito izvedbo kaskadne reakcije z in situ tvorbo H$_2$O$_2$ in primerljivo volumetrično produktivnostjo glede na proste encime. Mikropretočni sistem za pripravo CLEA-HRP-GOx odpira možnosti za nadaljnjo integracijo priprave in biotransformacije v kontinuirnem procesu.

• Language: Slovenian
• Keywords: imobilizacija encimov, hrenova peroksidaza, mikropretočni sistem, resveratrol, δ-viniferin
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2026
• PID: 20.500.12556/RUL-182923
• COBISS.SI-ID: 279677443
• Publication date in RUL: 28.05.2026

Secondary language

• Language: English
• Title: Preparation and application of combined cross-linked enzyme aggregates in microfluidic systems for the biocatalytic synthesis of δ-viniferin

Abstract

This master's thesis addresses the preparation and application of combined cross-linked enzyme aggregates (CLEAs) in a microfluidic system for the biocatalytic synthesis of δ-viniferin from resveratrol. The study is based on coupling the biocatalytic oxidation of resveratrol with H$_2$O$_2$, catalysed by horseradish peroxidase (HRP), with the in situ generation of this substrate through a reaction catalysed by glucose oxidase (GOx), thereby reducing the inhibitory effect of H$_2$O$_2$ on HRP. Resveratrol is a natural phenolic compound present in grapes and consequently also in grape pomace, which is generated as a by-product of wine production. Grape pomace represents a significant agro-industrial waste stream due to its content of biologically active compounds. Its utilisation as a raw material constitutes an example of waste valorisation, as it enables the conversion of a low-value residue into a value-added product such as δ-viniferin. Within the scope of this work, a microfluidic system was developed for the preparation of cross-linked enzyme aggregates of HRP (CLEA-HRP) and combined cross-linked enzyme aggregates of HRP and GOx (CLEA-HRP-GOx). The microfluidic approach enables precise control over precipitation and cross-linking conditions, resulting in a more uniform particle size distribution and improved retained enzymatic activity compared to conventional batch procedures. The preparation conditions were optimised with respect to residence time, type and concentration of precipitating and cross-linking agents, and buffer selection. The CLEA-HRP-GOx enzyme preparation was applied in the batch biotransformation of resveratrol, and the reaction progress was monitored using NMR spectroscopy. The activities of both enzymes in the cross-linked aggregates were compared with those of free enzymes. The results demonstrate that CLEA-HRP-GOx enable efficient cascade reactions with in situ H$_2$O$_2$ generation and comparable volumetric productivity relative to free enzymes. The microfluidic system developed for CLEA-HRP-GOx preparation provides opportunities for further integration of catalyst preparation and biotransformation into a continuous process.

• Keywords: enzyme immobilization, horseradish peroxidase, microfluidic system, resveratrol, δ-viniferin