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Glede na naraščajoči trend svetovne populacije, izčrpavanje fosilnih virov in glede na očitne podnebne spremembe, je razvoj zelenih in trajnostno naravnanih tehnologij čedalje bolj pomemben. Lignocelulozna biomasa je bogato prisoten obnovljiv vir iz katerega lahko proizvajamo vrsto platformskih kemikalij. Primer je 5-hidroksimetilfurfural (HMF), produkt dehidracije glukoze in fruktoze, ki ga pridobivamo z depolimerizacijo celuloze. HMF lahko preko tristopenjske oksidacije pretvorimo do 2,5- furandikarboksilne kisline (FDCA). Slednjo je Ameriški Sektor za Energijo (DOE) uvrstil na seznam 12 najpomembnejših platformskih kemikalij pridobljenih iz biomase. FDCA ima zaradi strukturne podobnosti s tereftalno kislino priložnost izpodriniti ogromen delež fosilno-osnovane polietilen-tereftalatne (PET) plastike in jo zamenjati z bioplastiko. Tristopenjsko oksidacijo HMF do FDCA lahko dosežemo po kemijski poti, vendar ta zahteva uporabo dragih katalizatorjev, visokega tlaka in temperature ter toksičnih organskih topil. V zadnjem času zasledimo porast raziskav usmerjenih v encimsko oksidacijo HMF do FDCA, ki je precej bolj ekološka, saj ta poteka pri sobnem tlaku in temperaturi. V magistrskem delu smo tristopenjsko oksidacijo HMF do FDCA razdelili na posamezne intermediate in testirati katalitično aktivnost šestih komercialno dostopnih encimov: alkohol oksidaze, galaktoze oksidaze, katalaze, lakaze, lignin peroksidaze in hrenove peroksidaze. Aktivnost encimov smo testirali na vse intermediate v omenjeni oksidacijski poti. Katalitične aktivnosti so bile nizke z izjemo, ko je bil substrat 5-formil- 2-furankarboksilna kislina (FFCA), ki je bila v nekaterih primerih pretvorjena v FDCA z izkoristki večjimi od 80 %. Določili smo tudi nekatere fizikalno-kemijske lastnosti kislinskih derivatov HMF, kjer smo ugotovili, da slednji v raztopinah tvorijo asociate. Kislinskim derivatom HMF smo na podlagi kvantnomehanskega pristopa izračunali elektronsko gostoto in določili njihove pKa vrednosti. With the rapid growth of the world population and continuing depletion of petroleum reserves, green approaches using renewable resources for the production of chemicals will be required. Lignocellulosic biomass is an abundant, inexpensive, and sustainable resource from which platform chemicals can be derived. 5-Hydroxymethylfurfural (HMF) is derived from cellulose via dehydration of glucose and fructose. HMF can be further converted to 2,5-furandicarboxylic acid (FDCA) through three-step oxidation. The latter is listed as one of twelve top biomass-derived value-added chemicals by the American Department of Energy (DOE). The most promising application of FDCA is the replacement of fossil-based terephthalic acid in synthesis of polyethylene terephthalate (PET) plastics. Three-step oxidation of HMF to FDCA can be achieved via chemical pathway, but it requires special catalysts, high pressure and temperature and organic solvents which make process environmentally unfriendly and costly. A lot of research has been recently pointed into enzymatic conversion of HMF to FDCA which, by contrast, requires mild conditions such as room pressure and temperature, producing less toxic waste and is thus eco-friendlier. In present master thesis we studied catalytic activity of six commercially available enzymes: alcohol oxidase, galactose oxidase, catalase, laccase, lignin peroxidase and horseradish peroxidase in field of oxidising HMF to FDCA. We broke down the aforementioned three-step oxidation to separate reactions to determine all the possible substrates as well as reaction products for each individual enzyme. The product yields obtained with these single-enzyme reactions were mostly low, except in case when the substrate was 5-formyl-2-furancarboxylic acid (FFCA), where some of the yields of the conversion of FFCA to FDCA exceeded 80 %. We have also determined some of the physical properties of the HMF acid derivatives, where we found that they form associates in solutions. Based on quantum mechanics approach we calculated electron density for each exanimated HMF acid derivative as well we determined corresponding pKa values.

V letu 2013 smo na Kmetijskem inštitutu Slovenije raziskovali vpliv organske spojine hydroxymethylfurfural (HMF) na dolgoživost delavk medonosne čebele Apis mellifera carnica v laboratorijskih pogojih. V raziskavi smo ugotovili, da zviševanje odmerka HMF-a, dodanega v osnovno čebeljo hrano (apifonda), ne kaže večja odstopanja v smrtnosti čebel do 15. dneva. Med 16. in 30. dnevom je smrtnost čebel postopoma naraščala glede na odmerke 100, 500, 1000 in 1500 mg HMF-a/kg apifonde, in sicer pri vsakem višjem odmerku je bila stopnja preživetja krajša. . Negativen vpliv HMF-a se kaže tudi po 30 dnevu, kjer je pri najvišjem odmerku HMF-a skupina čebel živela zgolj še par dni, pri vsakem nižjem odmerku je bila stopnja preživetja čebel višja, zadnje čebele v kontrolni skupini pa so dočakale tudi do 45 dni. Hkrati smo ugotavljali, ali obstajajo razlike med skupinami čebel glede na količino zaužite hrane pri odmerkih 100 in 1500 mg HMF-a/kg apifonde. Raziskava kaže, da v prvih 7. dneh statističnih razlik ni bilo (p=0,245), oz. da so čebele brez večjih odstopanj enakomerno uživale hrano ne glede na višino odmerka HMF-a, primešanega v apifondo. Prisotnost okužbe Nosema spp. je tekom prvega poskusa bila zaznana v precej razpršenih količinah, v ponovitvi je bila zaznana zgolj občasno, pri poletnih čebelah pa ni bila zaznana, pri čem je upad oz. izguba spor Nosema spp. najverjetneje posledica odmiranja zimskih čebel iz prvega poskusa. S tehniko TUNEL smo ugotavljali celično smrt čebel, tretiranih z HMF-om. Iz histološke analize je razvidno, da je med 5. in 9. dnem opažena stagnacija v številu pozitivnih jeder, zaznan je celo upad v skupini z najvišjim odmerkom. Pri odmerku 500 mg HMF-a/kg apifonde je pri vsakem vzorčenju zaznano precej konstantno, in sicer visoko št. pozitivnih celic. Vrh okužbe je zabeležen 15. dan raziskave, pozneje sta zaznana bodisi stagnacija ali upad, zlasti pri najvišjem odmerku. In 2013 we have conducted a research about the effects of the organic compound hydroxymethylfurfural (HMF) on the longevity of honey bee drones Apis mellifera carnica in laboratory conditions at the laboratories of the Kmetijski inštitut Slovenije. Our research showed that the increase HMF in the basic bee nourishment (apifond) does not reflect in increased fatalities of bees until the age of 15 days. Between days 16 and 30 the fatalities increase, with higher fatality trends corresponding to higher concentration of HMF in nourishment (100, 500, 1000, 1500 mg/kg of HMF). The negative effect of HMF is also noticeable after day 30. In the group receiving the highest concentration of HMS none of the bees survived for more than a few days past day 30, while there were bees surviving longer corresponding to the lower concentration of HMF in their nourishment. In the test groups there were surviving bees up to day 45. We have also tested if there are differences between groups of bees receiving the lowest (100mg/kg) and highest (1500mg/kg) concentrations of HMF in their nourishment. The results for the first seven days show no statistically significant differences (p=0,245), the bees were consuming nourishment (without HMF and with both concentrations) in comparable amounts. The presence of Nosema spores in the midgut of bees did not show a relation to the concentration of HMF in their nourishment. Using the imunohistological method (TUNEL) we have determined the cell death in the epithel of the midgut of the bees receiving HMF. There was a noticeable reduction of the apoptosis level in the epithel of the bees consuming the nourishment with the concentration of 1500mg/kg of HMF between days 5 and 9. An increase of necrotic deterioration of the cells was also noted. Throughout the experiment, a high amount of positive, apoptosic cells was noticed only in the group of bees nourished with the concentration of 500mg/kg of HMF. The bees nourished with the lowest concentration did not survive until day 30, indicating that the lowest concentration of HMF (100mg/kg) causes changes in the intensity of apoptosis, which is noticeable in comparison with bees receiving pure apifond without HMF. The highest mortality of bees is noticed in the period between day 15 and day 30 and was caused by the concentration of 1500mg/kg of HMF, while the cellular changes are noticed with the lowest concentration of 100mg/kg of HMF in apifond.