| Abstract | U posljednje vrijeme sve više raste interes za upotrebom enzima kao katalizatora. Pri tome se posebno ističe enzim lipaza kojega karakteriziraju velika aktivnost u blagim reakcijskim uvjetima i širok spektar reakcija koje katalizira. Zbog toga je od presudne važnosti proizvesti enzim lipaza velike katalitičke aktivnosti za što je neophodno provesti njegovo pročišćavanje. Pročišćavanje enzima je vrlo često skup i spor postupak ako se koriste konvencionalne metode pa se u današnje vrijeme teži razvoju alternativnih, održivih i učinkovitih metoda. Velik interes u kontekstu pročišćavanja proteina i drugih biomolekula privlači upotreba vodenih dvofaznih sustava koji su prepoznati kao zelena i jeftina ekstrakcijska sredstva, a dodatna prednost im je izražena fleksibilnost. Vodeni dvofazni sustavi na bazi eutektičnih otapala zbog svoje netoksičnosti i biorazgradivosti odgovaraju svim zahtjevima i principima zelene kemije. Za procese pročišćavanja osjetljivih biomolekula kao što su proteini i enzimi često se primjenjuju i mikrosustavi jer omogućuju intenzifikaciju procesa uz vrlo malu vjerojatnost narušavanja strukture molekula te provedbu kontinuiranog procesa. U ovom radu razvijena je metoda za kontinuiranu mikroekstrakciju proteina, odnosno pročišćavanje enzima lipaza korištenjem vodenog dvofaznog sustava na bazi eutektičnog otapala. Nakon ispitivanja utjecaja sastava eutektičnog otapala i koncentracije soli na učinkovitost ekstrakcije u šaržnom sustavu, optimalnim se pokazao sustav s eutektičnim otapalom betain: urea i koncentracijom K2HPO4 od 0,7 g/mL. Odabrani vodeni dvofazni sustav korišten je za ekstrakciju proteina u mikroekstraktoru gdje je ispitan utjecaj vremena zadržavanja, ultrazvuka, temperature i promjera kanala na učinkovitost procesa ekstrakcije. Optimalna učinkovitost ekstrakcije iznosila je 98,50 %, a ostvarena je za vrijeme zadržavanja od 30 s u mikrosustavu promjera kanala 1000 μm i pri temperaturi 25 °C. S obzirom na učinkovitost ekstrakcije u šaržnom sustavu koja je iznosila 94,70 % za vrijeme od 30 min primjena mikroekstraktora rezultirala je intenzifikacijom procesa. Ispitana je i mogućnost ponovne uporabe eutektičnog otapala te je ono uspješno korišteno u nekoliko ekstrakcijskih ciklusa u šaržnom sustavu i u mikrosustavu. Razvijena metoda mikroekstrakcije i kapacitet eutektičnog otapala dodatno su ispitani korištenjem sirovog enzima lipaza proizvedenog fermentacijom Thermomyces lanuginosus na pogači buče. Uz to, proces mikroekstrakcije je uspješno opisan 2D matematičkim modelom. |
| Abstract (english) | Recently, there has been a growing interest in the use of enzymes as catalysts. Lipases thereby especially stand out while they are characterized by high activity in mild reaction conditions and a wide spectrum of reactions they catalyse. Therefore, it is crucial to produce the lipase of high catalytic activity which is commonly achieved by its purification. Enzyme purification is very often an expensive and slow procedure when conventional methods are used, so nowadays alternative, sustainable, and effective methods are being developed. The use of aqueous two-phase systems, that are recognized as green and cheap extraction agents, attracts a great interest in the purification of proteins and other molecules. An additional advantage is their significant flexibility, while aqueous two-phase systems can also be based on deep eutectic solvents which meet all the requirements and green chemistry principles due to their nontoxicity and biodegradability. For purification of sensitive biomolecules, such as proteins and enzymes, microsystems often stand out because they enable process intensification with a negligible disruption of molecule structures and carrying out a continuous process. In this paper, a method for continuous protein microextraction, namely lipase purification using a deep eutectic solvent based aqueous two-phase system was developed. After analysing the influence of deep eutectic solvent composition and salt concentration on extraction efficiency in the batch system, the system with betaine: urea deep eutectic solvent and K2HPO4 concentration of 0,7 g/mL proved to be optimal. The selected aqueous two-phase system was used for protein extraction in a microextractor where the influence of retention time, ultrasound, temperature, and channel diameter on the extraction process efficiency was examined. The optimal extraction efficiency achieved was 98,50 % for 30 s in a microsystem with a channel diameter of 1000 μm at 25 °C. The process was significantly intensified using a microextractor considering the extraction efficiency in a batch system which was 94,70 % for 30 min. The deep eutectic solvent reuse was also investigated, and it was successfully used in several extraction cycles in the batch system as well as in the microsystem. The developed microextraction method and the deep eutectic solvent capacity were further analysed using crude lipase produced by fermentation of Thermomyces lanuginosus on pumpkin oil pomace. Additionally, the microextraction process was successfully described by a 2D mathematical model. |
