Imobilizacija amin transaminaze N-His$_6$-ATA-v1 na funkcionalizirane silikatne delce

Triler, Katja (author), Žnidaršič Plazl, Polona (mentor)

Encimi so zaradi svojih lastnosti potencialni katalizatorji za številne industrijske procese. Za povečanje stabilnosti encimov, njihovo enostavnejšo regeneracijo in v izogib dragim separacijskim postopkom se kot rešitev pojavljajo različni načini imobilizacije encimov. Amin transaminaze (ATA) imajo ključno vlogo pri prenosu aminskih skupin z donorja amina na akceptor amina in so zato zelo zanimive za uporabo predvsem v farmacevtski industriji. V sklopu magistrske naloge smo poskušali izbrani encim, amin transaminazo N-His$_6$-ATA-v1, imobilizirati na silikatne delce, da bi pridobili samosestavljive strukture za potencialno uporabo v mikroreaktorjih. Vezava encima na silikatne delce temelji na tvorbi koordinacijske vezi med heksahistidinskim (His$_6$) označevalcem encima in silikatnimi delci, ki so površinsko funkcionalizirani z različnimi alifatskimi verigami in kovinskimi ioni. Pri eksperimentalnem delu smo izbrani encim izrazili v bakterijah Escherichia coli in ga očistili s preparativno afinitetno kromatografijo. Po izražanju smo encim okarakterizirali. Na osnovi spektrofotometrije smo določili koncentracije encima, opredelili čistost encima na osnovi gelske elektroforeze in z encimskimi testi določili aktivnosti prostega encima. Encim smo nato imobilizirali na delce različnih velikosti (100, 250 in 500 nm) s tremi različnimi dolžinami alifatskih verig in različnimi, koordinacijsko vezanimi kovinskimi ioni (bakrovimi, kobaltovimi, gadolinijevimi in železovimi). Po inkubaciji encimov s funkcionaliziranimi delci smo imobiliziranim encimom določili aktivnost in z uporabo kazalcev uspešnosti imobilizacije encima (izkoristek imobilizacije, učinkovitost imobilizacije, zadržana aktivnost) ugotovili, kateri delci (glede na velikost in dolžino alifatskih verig ter vrsto kovinskega iona) so najbolj primeri za imobilizacijo izbrane amin transaminaze. Rezultati eksperimentalnega dela so pokazali, da je imobilizacija najbolj optimalna na najmanjših delcih z najdaljšo verigo in koordinacijsko vezanimi kobaltovimi ioni. Izkoristek imobilizacije je v optimalnem primeru 79,48 %, učinkovitost imobilizacije 46,35 % in zadržana aktivnost 36,83 %.

• Language: Slovenian
• Keywords: imobilizacija, amin transaminaza, funkcionalizirani delci, biotransformacija
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2023
• PID: 20.500.12556/RUL-153380
• COBISS.SI-ID: 181553411
• Publication date in RUL: 28.12.2023

Secondary language

• Language: English
• Title: Amine transaminase N-His$_6$-ATA-v1 immobilization on functionalized silica particles

Abstract

Enzymes possess unique properties that render them potential catalysts for a wide array of industrial processes. In the quest to bolster enzyme stability, simplify their regeneration, and circumvent expensive separation techniques, diverse approaches to enzyme immobilization have emerged as a compelling solution. Aminotransferases (ATAs) play a crucial role in the transfer of amino groups from amino donor to amino acceptor and are therefore highly interesting for use, especially in the pharmaceutical industry. In the context of this MSc thesis, we have attempted to immobilize a selected enzyme, amine transaminase N-His$_6$-ATA-v1, on silicate particles to obtain self-assembling structures for potential use in microreactors. The technique relied on establishing a coordination bond between the enzyme's hexahistidine (His$_6$) tag and silicate particles functionalized with various aliphatic chains and metal ions. In the experimental work, the selected enzyme was expressed in Escherichia coli and purified by preparative affinity chromatography. Post-expression, we characterized the enzyme by determining its concentration with spectrophotometry, assess its purity via gel electrophoresis and determined the activity of free enzymes with enzyme assays. Subsequently, the enzyme was immobilized onto particles of differing sizes (100, 250, and 500 nm) coupled with varying lengths of aliphatic chains and an array of coordination-bound metal ions (copper, cobalt, gadolinium, and iron). After incubation of the enzymes with the functionalized particles, we gauged the activity of the immobilized enzymes using metrics such as immobilization yield, efficiency, and retained activity. This evaluation aimed to pinpoint the most suitable nanoparticles (considering size, aliphatic chain length, and the type of metal ion) for immobilizing the selected amine transferase. The experimental findings suggest that the smallest particles with the longest chains and cobalt ions as the coordination entity are optimal for immobilization. In this scenario, the immobilization yield reached 79.48%, with immobilization efficiency of 46.35% and a retained activity of 36.83%

• Keywords: immobilization, aminotransferase, functionalized particles, biotransformation