Pročišćavanje enzima MenD za primjenu u biokatalizi

undergraduate thesis

• Title (english): Purification of the MenD enzyme for application in biocatalysis
• Author: Laura Glavinić
• Mentor: Martina Sudar (mentor)
• Committee member: Martina Sudar (predsjednik povjerenstva)
• Committee member: Zvjezdana Findrik Blažević (član povjerenstva)
• Committee member: Monika Šabić Runjavec (član povjerenstva)
• Granter: University of Zagreb; Faculty of Chemical Engineering and Technology; Zagreb
• Defense date and country: 2024-07-12, Croatia
• Scientific / art field, discipline and subdiscipline: TECHNICAL SCIENCES; Chemical Engineering

Abstract

U dobu zelene revolucije svijest o potrebi pronalaženja održivijih alternativa tradicionalnim industrijskim procesima, vrstama kemikalija i materijalima svakog je dana sve veća. Gotovo svi industrijski procesi koriste katalizatore koji su uglavnom štetni za okoliš, nerazgradivi i zahtijevaju vrlo oštre reakcijske uvjete. S druge strane, biokatalizatori su najčešće enzimi, molekule evoluirale u fiziološkom okruženju koje su u potpunosti biorazgradive, a opstaju u blagim reakcijskim uvjetima gdje pokazuju najveću aktivnost i učinkovitost. Sama biokataliza pronalazi svoje mjesto u gotovo svim načelima zelene kemije, a potencijal koji biokatalizatori pokazuju za zamjenu tradicionalnih kemijskih procesa, čineći ih ekološki prihvatljivijima eksponencijalno raste razvojem inovacija i tehnoloških napredaka posebno na područjima enzimsko-proteinskog i genetičkog inženjerstva. Enzim MenD, 2-sukcinil-5-enolpiruvil-6-hidroksi-3-cikloheksen-1-karboksilat (SEPHCHC)-sintaza ključan je u sintezi menakinona, ima potencijalno antibiotsko djelovanje u lijekovima protiv tuberkuloze, katalizira reakcije nastanka spojeva s 1,4-funkcionalnim skupinama i jedini je enzim za kojeg je poznato da katalizira reakciju adicije ThDP-a na β-ugljik sekundarnog supstrata. U ovom radu provedeno je pročišćavanje uzoraka proteina MenD, masene koncentracije 𝛾=41,67 mg/mL. Uzorak je pročišćen afinitetnom kromatografijom na uređaju ÄKTAprime plus, koristeći HisTrap™ excel kromatografsku kolonu. Ovakve kromatografske kolone omogućavaju hvatanje i pročišćavanje proteina označenih histidinom afinitetnom kromatografijom na imobiliziranim ionima metala. Uspješnost pročišćavanja provjerena je elektroforezom, kojom je ispitana prisutnost MenD-a u pročišćenim frakcijama.

Abstract (english)

In the era of green revolution, awareness of the need to find sustainable alternatives to traditional industrial processes, chemicals and materials grows greater every day. Nearly every industrial process uses catalysts that are harmful to the environment, nondegradable and require very harsh reaction conditions. On the other hand, biocatalysts are mostly enzymes, molecules evolved in a physiological environment that are fully biodegradable and survive in mild reaction conditions where they show the greatest activity and efficiency. Biocatalysis follows almost every green chemistry principle and the potential that biocatalysts have regarding replacing traditional chemical processes, making them more environmentally friendly, grows exponentially with the development of innovations and technological advancements especially in areas of enzymatic and genetic engineering. MenD is the designation for the enzyme 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate (SEPHCHC) synthase, which is important in the synthesis of menaquinone, has potential antibiotic properties in anti-tuberculosis medicines, catalyses reactions that give
1,4-functionalized products and is the only known enzyme that catalyses the addition of ThDP on β-carbon of a secondary substrate. Purification of protein sample containing MenD, mass concentration of γ=41,67 mg/mL, was performed in this work. The sample was purified by affinity chromatography on ÄKTAprime plus via HisTrap™ excel chromatography column. These chromatography columns enable the capture and purification of histidine labelled proteins by immobilized metal affinity chromatography. The success of the purification was determined using electrophoresis, which tested the presence of MenD in purified fractions.

• Keywords: biokataliza; biokatalizatori; MenD; pročišćavanje proteina; afinitetna kromatografija
• Keywords (english): biocatalysis; biocatalysts; MenD; protein purification; affinity chromatography
• Language: croatian
• URN:NBN: urn:nbn:hr:149:515108
• Study programme: Title: Chemical Engineering - Undergraduate study; Study programme type: university; Study level: undergraduate; Academic / professional title: sveučilišni/a prvostupnik/prvostupnica (baccalaureus/baccalaurea) inženjer/inženjerka kemijskog inženjerstva (sveučilišni/a prvostupnik/prvostupnica (baccalaureus/baccalaurea) inženjer/inženjerka kemijskog inženjerstva)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Embargoed access; Embargo expiration date: 2026-12-31
• Created on: 2024-07-19 13:18:06