Imobilizacija amin transaminaze na funkcionalizirane silikatne nanodelce preko heksahistidinskega označevalca

Omerzel, Sara (author), Žnidaršič Plazl, Polona (mentor)

Encimi so beljakovine, ki delujejo kot biološki katalizatorji. Delujejo pri blagih pogojih, so specifični za določen substrat in enantioselektivni. V industriji se spopadamo z izzivom slabe stabilnosti encimov pri grobih industrijskih pogojih, zato jih pogosto imobiliziramo. Z imobilizacijo preprečimo izgube encimov, ki so pogoste pri recikliranju prostih encimov, izboljša se njihova stabilnost pri procesnih pogojih, s tem pa omogočamo njihovo dolgotrajno uporabo v kontinuirnih procesih, ki so trend razvoja sodobne trajnostne proizvodnje snovi. Amin transaminaze so encimi, ki katalizirajo prenos aminske skupine iz donorja na akceptor amina ob prisotnosti koencima piridoksal-5'-fosfat. Encim N-His6-ATA-wt, ki je označen s heksahistidinskim (His6) označevalcem, smo uspešno izrazili in ga imobilizirali na funkcionalizirane silikatne nanodelce, s čimer smo dosegli tvorbo samosestavljivih struktur. Najprej smo silikatne nanodelce funkcionalizirali z aminosilanskimi verigami in jih nato kompleksirali z različnimi kovinskimi ioni. Sledila je imobilizacija encima, ki se preko heksahistidinskega označevalca koordinativno veže na funkcionalizirane silikatne nanodelce. Imobilizacijo N-His6-ATA-wt smo izvedli na različnih velikostih silikatnih nanodelcev (povprečni premeri 100, 250 oz. 500 nm), z različnimi dolžinami aminosilanskih verig med nanodelcem in kovino in različnimi kovinskimi ioni (Cu2+, Ni2+, La3+). Uspešnost imobilizacije smo sledili z izračunom izkoristka in učinkovitosti imobilizacije ter zadržane aktivnosti. Glede na pridobljene rezultate smo ugotovili, da je za imobilizacijo N-His6-ATA-wt najbolj primeren silikatni nanodelec s povprečnim premerom 250 nm, z najdaljšo aminosilansko verigo in je kompleksiran z bakrovim ionom (Cu2+). Pri tem je izkoristek imobilizacije znašal 68,27 %, učinkovitost imobilizacije je bila 38,74 % in zadržana aktivnost 26,01 %. Zaznali smo trend, da je imobilizacija na manjše nanodelce z daljšimi aminosilanskimi verigami uspešnejša. 100 nm nanodelci temu trendu ne sledijo, kar bi lahko pripisali drugačnemu postopku funkcionalizacije.

• Language: Slovenian
• Keywords: amin transaminaza, biotransformacija, imobilizacija, silikatni nanodelci
• 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-152574
• COBISS.SI-ID: 176617987
• Publication date in RUL: 29.11.2023

Secondary language

• Language: English
• Title: Immobilization of amine transaminase on functionalized silicate nanoparticles via hexahistidine tag

Abstract

Enzymes are proteins that act as biological catalysts. They function under mild conditions, are substrate specific and enantioselective. In industrial production, we face the challenge of poor stability when enzymes are exposed to harsh industrial conditions and because of that, they are often immobilized. Immobilization prevents enzyme losses that are common when enzymes are recycled. Immobilization also improves enzyme stability under process conditions, which enables their long-term use in continuum processes. These processes are the trend in the development of modern sustainable production. Amine transaminases (ATAs) are enzymes that catalyze the transfer of an amine group from a donor to an acceptor of amine in the presence of the coenzyme pyridoxal 5’ phosphate (PLP). Enzyme N-His6-ATA-wt with hexahistidine (His6) tag was successfully expressed and immobilized on functionalized silica nanoparticles, resulting in the formation of self-assembled structures. The silica nanoparticles were first functionalized with aminosilane chains and then complexed with metal ions. This was followed by the immobilization of the free enzyme. Coordinative binding interaction was formed between the surface-funcionalized silica nanoparticles and the His6 tag of the enzyme. Immobilization of N-His6-ATA-wt was performed on different sizes of silica nanoparticles (average diameters of 100, 250, or 500 nm), with different aminosilane chains between the nanoparticle and the metal ion, and different metal ions (Cu2+, Ni2+, La3+). Immobilization was evaluated by immobilization yield and efficiency, and recovered activity. According to the obtained results, we found that the most suitable silica nanoparticles for the immobilization of N-His6-ATA-wt are those with the diameter of 250 nm with the longest aminosilane chain and in a complex with the copper ion (Cu2+). In this case, the immobilization yield was 68,27 %, the immobilization efficiency was 38,74 %, and recovered activity was 26,01 %. Our findings indicate that immobilization on smaller nanoparticles with longer aminosilane chains is more successful. The 100 nm nanoparticles do not follow this trend, presumably due to the difference in functionalization protocol.

• Keywords: amine transaminase, biotransformation, immobilization, silica nanoparticles