Title Razvoj integriranog mikrosustava za kontinuiranu ekstrakciju enzima lakaza Title (english) Development of an integrated microsystem for the continuous extraction of the enzyme laccase Author Marko Božinović Mentor Anita Šalić (mentor)Committee member Anita Šalić (predsjednik povjerenstva)Committee member Domagoj Vrsaljko (član povjerenstva)Committee member Martina Sudar (član povjerenstva)Granter University of Zagreb Defense date and country 2023-02-24, Croatia Scientific / art field, TECHNICAL SCIENCES Abstract Pročišćavanje enzima dugotrajan je, skup i zahtjevan proces koji najčešće rezultira s niskim prinosom i gubitkom aktivnosti enzima. Ekstrakcijske metode su među najraširenijim metodama pročišćavanja različitih molekula, međutim upotreba konvencionalnih otapala, kao što su organska otapala, dovodi do denaturacije enzima. Upotreba vodenih dvofaznih sustava u svrhu pročišćavanja biomolekula, pokazala se učinkovitom i ekološki prihvatljivom metodom. Između različitih vodenih dvofaznih sustava, posebnu pažnju privlače vodeni dvofazni sustavi na bazi eutektičnih otapala (engl. Deep Eutectic Solvents, DES) zbog svoje niske toksičnosti, visoke biorazgradivosti i ekonomičnosti. Posljednjih godina, pozornost se usmjerava i na upotrebi mikroekstraktora u svrhu pročišćavanje biomolekula zbog intenzifikacije proces i manje vjerojatnosti narušavanje strukture biomolekula. U ovom radu razvijen je integrirani sustav serijski povezanog mikroekstraktora i mikroseparatora za kontinuiranu mikroekstrakciju proteina, odnosno ekstrakciju enzima lakaza porijeklom iz Trametes versicolor korištenjem vodenog dvofaznog sustava na bazi DES-a. Ukupno je sintetizirano i karakterizirano 15 DES-ova. U seriji šaržnih ekstrakcija ispitan je utjecaj sastava DES-ova i koncentracije soli (K2HPO4) na učinkovitost ekstrakcije enzima lakaza. Sustav koji se sastojao od eutektičnog otapala kolin-klorid:propilen-glikol:cinkov diklorid 1:4:0,02 (ChCl:PG:ZnCl2 1:4:0,02) i vodene otopine K2HPO4 koncentracije γ = 0,5 g/mL pokazao se najučinkovitijim sustavom za ekstrakciju enzima lakaza. U sljedećem koraku, korištenjem odabranog sustava, ispitan je utjecaj koncentracije enzima na učinkovitost ekstrakcije, a dobiveni rezultati pokazali su da povećanje koncentracije enzima pozitivno utječe na učinkovitost ekstrakcije. U svrhu intenzifikacije procesa, ekstrakcija je provedena u mikroekstraktoru gdje su ispitani utjecaj promjera kanala i vremena zadržavanja na učinkovitost ekstrakcije. Najveća učinkovitost ekstrakcije enzima u mikroekstraktoru (d = 500 μm) iznosila je E = 88,34 ± 1,04 %, a ostvarena je za vrijeme zadržavanja od τ = 12 s. Proces ekstrakcije u mikroekstraktoru uspješno je opisan 2D matematičkim modelom. Nakon optimizacije procesa ekstrakcije, mikroekstraktor je serijski spojen s mikroseparatorom u kojem je provedena uspješna separacija ekstraktne i rafinatne faze. Na samom kraju, razvijena metoda ispitana je na sirovom enzimu lakaza proizvedenog fermentacijom Trametes versicolor, a ekstrahirani enzim upotrjebljen je za uklanjanje tekstilne boje na bazi anilina.
Abstract (english) Enzyme purification is a lengthy, expensive, and demanding process that usually results in low yield and loss of enzyme activity. Extraction procedures are among the most widely used methods for purifying various molecules. However, the use of conventional solvents, such as organic solvents, leads to denaturation of enzymes. The use of aqueous two-phase systems (ATPS) to purify biomolecules has been shown to be an effective and environmentally friendly method. Among the various ATPS, ATPS based on deep eutectic solvents (DES) are attracting special attention due to their low toxicity, high biodegradability, and cost-effectiveness. In recent years, attention has also focused on the use of microextractors for biomolecule purification because they intensify the process and are less likely to disrupt the structure of biomolecules. In this work, an integrated system of microextractor and microseparator connected in series was developed for continuous microextraction of proteins, i.e., extraction of the enzyme laccase from Trametes versicolor, using an ATPS based on DES. A total of 15 DESs were synthesized and characterized. In a series of batch extractions, the influence of DES composition and salt concentration (K2HPO4) on the efficiency of laccase enzyme extraction was investigated. The system consisting of the eutectic solvent choline-chloride:propylene glycol:zinc dichloride 1:4:0.02 (ChCl:PG:ZnCl2 1:4:0.02) and an aqueous solution of K2HPO4 with a concentration of γ = 0.5 g/mL proved to be the most efficient system for laccase enzyme extraction. In the next step, the influence of enzyme concentration on extraction efficiency was tested using the selected system. The obtained results showed that increasing the enzyme concentration positively affected the extraction efficiency. To intensify the process, the extraction was performed in a microextractor, and the influence of the channel diameter and residence time on the extraction efficiency was studied. The highest efficiency of enzyme extraction in the microextractor (d = 500 μm) was E = 88.34 ± 1.04 % and it was achieved at a residence time of τ = 12 s. The extraction process in the microextractor was successfully described by a 2D mathematical model. After optimization of the extraction process, the microextractor was connected in series with a microseparator, in which the extract and raffinate phases were successfully separated. At the very end, the developed method was tested on the crude laccase enzyme produced by the fermentation of Trametes versicolor, and the extracted enzyme was used to remove the aniline-based textile dye.
Keywords
enzim lakaza
ekstrakcija
eutektična otapala
mikroekstraktor
mikroseparator
integrirani sustav
Keywords (english)
enzyme laccase
extraction
deep eutectic solvents
microextractor
microseparator
integrated system
Language croatian URN:NBN urn:nbn:hr:149:829500 Study programme Title: Chemical Engineering - Graduate study Type of resource Text File origin Born digital Access conditions Open access Terms of use Created on 2023-03-10 13:22:12
