Izolacija i pročišćavanje halogenhidrin-dehalogenaze iz stanica genetski modificirane Escherichia coli

undergraduate thesis

• Title (english): Isolation and purification of halohydrin dehalogenase from the genetically modified Escherichia coli
• Author: Iva Mihaljević
• Mentor: Zvjezdana Findrik Blažević (mentor)
• Committee member: Zvjezdana Findrik Blažević (predsjednik povjerenstva)
• Committee member: Martina Sudar (član povjerenstva)
• Committee member: Marija Vuković Domanovac (član povjerenstva)
• Granter: University of Zagreb; Faculty of Chemical Engineering and Technology; Zagreb
• Defense date and country: 2020-09-22, Croatia
• Scientific / art field, discipline and subdiscipline: TECHNICAL SCIENCES; Chemical Engineering

Abstract

U ovom radu provedena je izolacija i pročišćavanje enzima halogenhidrin-dehalogenaze (HheC). Najprije je uzgojena biomasa genetski modificiranih stanica Escherichia coli s visokim udjelom HheC. Nakon razbijanja bakterijskih stanica ultrazvukom, odvojeni su proteini od ostatka staničnog materijala centrifugiranjem te je dobiven sirovi enzimski ekstrakt. Kako bi se utvrdila uspješnost eksperimenta, mjerena je koncentracija proteina metodom po Bradfordu i aktivnost enzima HheC spektrofotometrijskim testom. Proces izolacije HheC je bitan za biokatalizu jer je otkriveno da ovaj enzim ima sposobnost kataliziranja reakcije otvaranja epoksida s malim “neprirodnim” anionskim nukleofilima, kao što su azid, cijanid, cijanat, tiocijanat, nitrit i formijat. Također, bitna primjena HHDH enzima je u proizvodnji optički čistih C3 ili C4 prekursora, na primjer, (R)-4-cijano-3-hidroksibutanoata za sintezu statina.

Abstract (english)

In this work, isolation and purification of the enzyme halohydrin dehalogenase (HheC) were carried out. Firstly, biomass of genetically modified Escherichia coli cells with a high HheC percentage was grown. After the disruption of bacterial cells by ultrasound, the proteins were separated from the rest of the cellular material by centrifugation and the crude enzyme extract was obtained. To determine the success of the experiment, the Bradford method for protein quantification and the spectrophotometric assay for HheC enzyme activity were used. The HheC isolation process is important for biocatalysis because it has been found that this enzyme has the ability to catalyze the epoxide ring-opening reaction with small "unnatural" anionic nucleophiles such as azide, cyanide, cyanate, thiocyanate, nitrite and formate. Also, the essential application of HHDH enzymes is in the production of optically pure C3 or C4 precursors, for example, (R)-4-cyano-3-hydroxybutanoate for statin synthesis.

• Keywords: halogenhidrin-dehalogenaza; enzim; epoksid; izolacija enzima; statin
• Keywords (english): halohydrin dehalogenase; enzyme; epoxide; enzyme isolation; statin
• Language: croatian
• URN:NBN: urn:nbn:hr:149:702999
• Project: Number: IP-2018-01-4493; Title: Enzimska sinteza fluoriranih kiralnih građevnih blokova; Acronym: EnzyFluor; Leader: Maja Majerić-Elenkov; Jurisdiction: Croatia; Funder: HRZZ; Funding stream: IP
• Study programme: Title: Applied Chemistry - Undergraduate study; specializations in: Environmental chemistry and green technologies, Advanced materials and technologies, Applied organic chemistry; Course: Environmental chemistry and green technologies; Study programme type: university; Study level: undergraduate; Academic / professional title: sveučilišni/a prvostupnik/prvostupnica (baccalaureus/baccalaurea) primijenjene kemije (sveučilišni/a prvostupnik/prvostupnica (baccalaureus/baccalaurea) primijenjene kemije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access; Embargo expiration date: 2022-10-01
• Created on: 2023-05-05 12:04:58