Ispitivanje utjecaja organskih otapala na aktivnost halogenhidrin-dehalogenaze u reakciji otvaranja para-nitrostiren oksida

master's thesis

• Title (english): Investigation of the influence of organic solvents on the activity of halohydrin dehalogenase in the ring-opening reaction of para-nitrostyrene oxide
• Author: Magdalena Kolić
• 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: 2023-09-08, Croatia
• Scientific / art field, discipline and subdiscipline: TECHNICAL SCIENCES; Chemical Engineering

Abstract

Organska otapala se zbog svoje prirode i kompatibilnosti s nepolarnim organskim spojevima koriste u većini sintetskih reakcija u industrijskom mjerilu. Takvi kemijski procesi često zahtijevaju reakcije u više stupnjeva i kontaminirajuće kemijske reagense. Biotehnološka alternativa takvim kemijskim procesima uključuje enzime koji djeluju kao biokatalizatori, omogućuju korištenje sirovina na biološkoj osnovi te smanjuju broj koraka reakcije, što može biti popraćeno jeftinijom proizvodnjom. Kao rezultat ovog pristupa, dobivaju se produkti visoke kvalitete na ekonomski i tehnološki konkurentan i održiv način. Iako se biokatalitički procesi smatraju idealnim rješenjem za razvoj održivih proizvodnih sustava i unatoč ogromnom napretku u otkrivanju i razvoju enzima u području molekularne biologije, često postignuta niska produktivnost biokatalitičkog procesa, zajedno s niskom stabilnošću i aktivnosti enzima, sprječava da se njihov potencijal ostvari u industrijskim procesima. Halogenhidrin-dehalogenaze (HHDH) su vrlo rijetki enzimi koji prirodno sudjeluju u bakterijskoj razgradnji halogenih spojeva, a zbog njihovog katalitičkog potencijala pripadaju u skupinu industrijski relevantnih biokatalizatora. U radu je korišten C-tip enzima iz skupine halogenhidrin-dehalogenaza (HheC) izoliran iz genetski modificirane bakterije Escherichia coli. Ispitana je aktivnost HheC enzima, u prisutnosti dvanaest različitih organskih otapala, testom pomoću para-nitrostiren oksida (PNSO) s bromidnim ionima u reakciji otvaranja prstena uz nastajanje para-nitro-2-brom-1-feniletanola (PNSHH). Aktivnost enzima određena je metodom početnih reakcijskih brzina, a koncentracije PNSHH i PNSO određene su pomoću pripremljenih baždarnih pravaca. Pripremljeni uzorci su analizirani pomoću HPLC uređaja, a utjecaj volumnih udjela organskih otapala na specifičnu aktivnost enzima prikazan je grafički. Dobivene vrijednosti logP i c50 organskih otapala prikazane su tablično. Zbog toksičnosti ili zapaljivosti mnogih organskih otapala, sve više se koriste „zelene“ alternative u pogledu „zelenih“ organskih otapala. U radu su ispitana četiri „zelena“ organska otapala koja se dobivaju iz prirodnih izvora.

Abstract (english)

Due to their nature and compatibility with non-polar organic compounds, organic solvents are used in most synthetic reactions on an industrial scale. Such chemical processes often require multistep reactions and contaminating chemical reagents. A biotechnological alternative to such chemical processes includes enzymes that act as biocatalysts, enable the use of biologically based raw materials and reduce the number of reaction steps which can be accompanied by cheaper production. As a result, high quality products are obtained in an economically and technologically competitive and sustainable manner. Although biocatalytic processes are considered as an ideal solution for the development of sustainable production systems and despite tremendous advances in the discovery and development of enzymes in the field of molecular biology, the often achieved low productivity of the biocatalytic process together with the low stability and activity of enzymes, prevents their potential from being realized in industrial applications processes. Halohydrin dehalogenases (HHDH) are very rare enzymes that naturally participate in the bacterial degradation of halogenated compounds and due to their catalytic potential they belong to industrially relevant biocatalysts. This work uses a C-type enzyme from the halohydrin dehalogenase group (HheC) isolated from a genetically modified bacterium Escherichia coli. The activity of the HheC enzyme, in the presence of twelve different organic solvents, was tested using para-nitrostyrene oxide (PNSO) with bromide ions in the ring-opening reaction with the formation of para-nitro-2-bromo-1-phenylethanol (PNSHH). Enzyme activity was determined using the initial reaction rates method and the concentrations of PNSHH and PNSO were determined using a prepared calibration curves. The prepared samples were analyzed using an HPLC device and the influence of volume ratios of organic solvents on the specific activity of the enzyme was shown graphically. The obtained values of logP and c50 of organic solvents are presented in a table. Due to the toxicity or flammability of many organic solvents, there are green alternatives in terms of green organic solvents that are increasingly being used.
Four green organic solvents obtained from natural sources were tested in this paper.

• Keywords: organska otapala; biokataliza; halogenhidrin-dehalogenaze; „zelena“ organska otapala
• Keywords (english): organic solvents; biocatalysis; halohydrin dehalogenases; green organic solvents
• Language: croatian
• URN:NBN: urn:nbn:hr:149:570313
• 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: Environmental Engineering - Graduate study; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra inženjer/inženjerka ekoinženjerstva (magistar/magistra inženjer/inženjerka ekoinženjerstva)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2023-09-14 14:08:33