1. Enzyme activity and physiochemical properties of flour after supercritical carbon dioxide processingMaja Leitgeb, Željko Knez, Gordana Hojnik Podrepšek, 2022, original scientific article Abstract: The objectives of this study were to inactivate the enzymes α-amylase, lipase, protease,
and peroxidase in flour with supercritical carbon dioxide (scCO2), and to optimize the enzymatic
treatment conditions. Enzyme inactivation is important, due to the undesirability of certain flour
enzymes that cause adverse reactions during storage as unpleasant rancidity of flour, and, at the
same time, reduce the shelf life of flour. Therefore, crude enzymes and flour were initially exposed
to scCO2 to determine the effect on specific enzyme activity under appropriate conditions. The
activity of the unwanted enzymes lipase and peroxidase decreased under optimal process conditions
of scCO2 exposure, lipase by 30%, and peroxidase by 12%, respectively. It was discovered that the
inactivation of enzymes in wheat flour occurred, where, at the same time, this sustainable method
allows the regulation of enzyme activity in the baking process. Afterwards, the effect of scCO2 on the
physicochemical properties of flour, morphological changes on starch granules, and content of total
lipids was studied. In scCO2-treated white wheat flour, the fat content decreased by 46.15 ± 0.5%,
the grain structure was not damaged, and the bread as the final product had a lower specific surface
volume. Therefore, this could be a promising technology for flour pretreatment, potentially impacting
the prolonging of its shelf-life. Keywords: enzymes, enzyme activity, proteins, supercritical fluids, wheat flour Published in DKUM: 10.04.2025; Views: 0; Downloads: 1
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2. Efficient β-galactosidase immobilised on glycidyl methacrylate polyHIPEMuzafera Paljevac, Darja Pečar, Peter Krajnc, 2025, original scientific article Abstract: Poly(glycidyl methacrylate-co-ethyleneglycol dimethacrylate) monoliths (PolyGMA) were synthesized by high internal phase emulsion (HIPE) templating and polymerisation. The porous monoliths exhibited a hierarchical porous structure with primary pores (cavities, average diameter 35 µm) interconnected by secondary pores (average diameter 5 µm). FTIR spectroscopy confirmed the chemical composition and identified characteristic functional groups of both GMA and EGDMA. The polyGMA materials were ground and sieved to obtain particles between 710 µm and 1000 µm in diameter, which were subsequently used to immobilize the enzyme β-galactosidase. Immobilization was performed using two methods, namely direct binding via epoxide groups and binding after the activation with glutaraldehyde. The glutaraldehyde method resulted in higher enzyme loading (0.43 mg of enzyme per 100 mg of polyGMA) and significantly improved catalytic activity compared to direct binding. The immobilized β-galactosidase was used for lactose hydrolysis under various conditions using both batch and flow-through reactors. Optimal activity was observed at pH 6.5 and 35°C, with kinetic parameters vmax = 0.64 mmol∙L -1 ∙min-1 and �� = 38.8 mmol∙mol-1 . Reuse tests showed stable performance over five cycles. Comparatively, non-porous polyGMA exhibited negligible enzymatic activity compared to polyHIPE supported enzyme. In addition, lactose hydrolysis was investigated in a flow-through system at different flow rates (0.5–2.5 mL∙min- ¹). The highest conversion (100%) was observed at a flow rate of 0.5 mL∙min-¹, while a higher flow rate of 2.5 mL∙min-¹ resulted in a lower conversion (approx. 35%), both at the lactose concentration of 4 g∙L - ¹. Keywords: immobilized enzymes, glycidyl methacrylate, polyHIPE, β-galactosidase, beta-galactosidase Published in DKUM: 21.03.2025; Views: 0; Downloads: 4
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3. Mango peels as an industrial by-product: a sustainable source of compounds with antioxidant, enzymatic, and antimicrobial activityNika Kučuk, Mateja Primožič, Petra Kotnik, Željko Knez, Maja Leitgeb, 2024, original scientific article Abstract: Plant waste materials are important sources of bioactive compounds with remarkable health-promoting benefits. In particular, industrial by-products such as mango peels are sustainable sources of bioactive substances, with antioxidant, enzymatic, and antimicrobial activity. Appropriate processing is essential to obtain highly bioactive compounds for further use in generating value-added products for the food industry. The objective of the study was to investigate and compare the biological activity of compounds from fresh and dried mango peels obtained by different conventional methods and unconventional extraction methods using supercritical fluids (SFE). The highest total phenolic content (25.0 mg GAE/g DW) and the total content of eight phenolic compounds (829.92 µg/g DW) determined by LC-MS/MS were detected in dried mango peel extract obtained by the Soxhlet process (SE). SFE gave the highest content of proanthocyanidins (0.4 mg PAC/g DW). The ethanolic ultrasonic process (UAE) provided the highest antioxidant activity of the product (82.4%) using DPPH radical scavenging activity and total protein content (2.95 mg protein/g DW). Overall, the dried mango peels were richer in bioactive compounds (caffeic acid, chlorogenic acid, gallic acid, catechin, and hesperidin/neohesperidin), indicating successful preservation during air drying. Furthermore, outstanding polyphenol oxidase, superoxide dismutase (SOD), and lipase activities were detected in mango peel extracts. This is the first study in which remarkable antibacterial activities against the growth of Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) and Gram-positive bacteria (Bacillus cereus and Staphylococcus aureus) were evaluated by determining the microbial growth inhibition rate after 12 and 24 h incubation periods for mango peel extracts obtained by different methods. Ethanolic SE and UAE extracts from dried mango peels resulted in the lowest minimum inhibitory concentrations (MIC90) for all bacterial species tested. Mango peels are remarkable waste products that could contribute to the sustainable development of exceptional products with high-added value for various applications, especially as dietary supplements. Keywords: mangifera indica, peels, bioactive substances, LC-MS/MS, proteins, enzymes, antibacterial activity Published in DKUM: 12.08.2024; Views: 104; Downloads: 10
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4. Self-organization of enzyme-catalyzed reactions studied by the maximum entropy production principleAndrej Dobovišek, Marko Vitas, Tina Blaževič, Rene Markovič, Marko Marhl, Aleš Fajmut, 2023, original scientific article Abstract: The self-organization of open reaction systems is closely related to specific mechanisms that allow the export of internally generated entropy from systems to their environment. According to the second law of thermodynamics, systems with effective entropy export to the environment are better internally organized. Therefore, they are in thermodynamic states with low entropy. In this context, we study how self-organization in enzymatic reactions depends on their kinetic reaction mechanisms. Enzymatic reactions in an open system are considered to operate in a non-equilibrium steady state, which is achieved by satisfying the principle of maximum entropy production (MEPP). The latter is a general theoretical framework for our theoretical analysis. Detailed theoretical studies and comparisons of the linear irreversible kinetic schemes of an enzyme reaction in two and three states are performed. In both cases, in the optimal and statistically most probable thermodynamic steady state, a diffusion-limited flux is predicted by MEPP. Several thermodynamic quantities and enzymatic kinetic parameters, such as the entropy production rate, the Shannon information entropy, reaction stability, sensitivity, and specificity constants, are predicted. Our results show that the optimal enzyme performance may strongly depend on the number of reaction steps when linear reaction mechanisms are considered. Simple reaction mechanisms with a smaller number of intermediate reaction steps could be better organized internally and could allow fast and stable catalysis. These could be features of the evolutionary mechanisms of highly specialized enzymes. Keywords: enzymes, kinetic data analysis, steady state, self-organization, maximum entropy production Published in DKUM: 08.05.2024; Views: 234; Downloads: 11
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5. Biochemical characterization of black and green mutant elderberry during fruit ripeningMaja Mikulič Petkovšek, Anton Ivančič, Saša Gačnik, Robert Veberič, Metka Hudina, Silvija Marinovic, Christian Molitor, Heidi Halbwirth, 2023, original scientific article Abstract: The content of sugars, organic acids, phenolic compounds and selected enzyme activities in the anthocyanin pathway were analyzed in NIGRA (Sambucus nigra var. nigra—black fruits) and VIRIDIS (S. nigra var. viridis—green fruits) fruits over four stages of ripening. The share of glucose and fructose in green fruits was higher than in colored fruits, and the sugar content increased significantly until the third developmental stage. Ripe NIGRA berries had 47% flavonol glycosides, 34% anthocyanins, 3% hydroxycinnamic acids and 14% flavanols, whereas the major phenolic group in the VIRIDIS fruits, making up 88% of the total analyzed polyphenols, was flavonols. NIGRA fruits were rich in anthocyanins (6020 [mi]g g$^{-1}$ FW), showing strong activation of the late anthocyanin pathway (dihydroflavonol 4-reductase, anthocyanidin synthase). In both color types, phenylalanine ammonia lyase and chalcone synthase/chalcone isomerase activities were highest in the first stage and decreased during ripening. In VIRIDIS fruit, no anthocyanins and only one flavanol (procyanidin dimer) were found. This was most likely caused by a lack of induction of the late anthocyanin pathway in the last period of fruit ripening. The VIRIDIS genotype may be useful in studying the regulatory structures of anthocyanin biosynthesis and the contribution of distinct flavonoid classes to the health benefits of elderberries. Keywords: black elderberry, Sambucus nigra, green genotype, polyphenolics, sugars, organic acids, phenylpropanoid enzymes Published in DKUM: 28.03.2024; Views: 208; Downloads: 21
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6. (Magnetic) cross-linked enzyme aggregates of cellulase from T. Reesei: a stable and efficient biocatalystDušica Ifko, Katja Vasić, Željko Knez, Maja Leitgeb, 2023, original scientific article Keywords: immobilized catalysts, biocatalysis, enzymes, cellulase activity, CLEAs, mCLEAs, optimization, characterization, parameters Published in DKUM: 06.12.2023; Views: 501; Downloads: 171
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7. Production of enzymes from medicinal mushrooms : master's thesisGonzalo Herranz Gómez, 2022, master's thesis Abstract: The purpose of this master's thesis was to determine the ability of certain organisms of the kingdom Fungi to produce a series of enzymes in their active form by solid-state fermentation. In this study, two types of fungi were used, Pleurotus ostreatus and Ganoderma lucidum. For this purpose, different growth media, cultivation times (8 and 10 days), extraction procedures (shaking and homogenization) and extraction medium (distilled water, sodium citrate buffer and sodium phosphate buffer) were used.
First, for P. ostreatus mushroom, the optimization of the extraction procedure and time for isolation of enzymes in their active form (α-amylase, glucoamylase, cellulase, laccase, and protease) was studied. It was observed that the highest total protein concentration in mycelium extract was obtained by 8 min of homogenization (0.8607 mg/mL, and distilled water). Using the shaking procedure, the highest enzyme activities were achieved for α-amylase (24 h, 8.0413 U/mL, and sodium citrate buffer) and protease (3 h, 0.0040 U/mL, and sodium citrate buffer). With the homogenization process, the highest activities were achieved for the enzymes glucoamylase (10 min, 6.7113 U/mL, and sodium citrate buffer) and laccase (8 min, 12.2500 U/mL, and sodium citate buffer).
For the mushroom G. lucidum, the growth medium and the extraction procedure were optimized, using the same extraction medium (sodium citrate buffer). In this case, α-amylase, glucoamylase, cellulase, laccase, protease, catalase, peroxidase, superoxidase dismutase (SOD), and lipase were studied. It was observed that the highest total protein concentration was obtained with 4 min of homogenization (0.0338 mg/mL). Furthermore, using the homogenization process, the highest activities were achieved for α-amylase (4 min, 16.3459 U/mL) and SOD (4 min, 9.2615 U/mL). With the shaking procedure, the highest activities were achieved for cellulase (3 h, 1.6332 U/mL), lipase (3 h, 16.924 U/mL), glucoamylase (3 h, 14.6737 U/mL), peroxidase (3 h, 0.0156 U/mL), protease (3 h, 0.0080 U/mL) and laccase (24 h, 20.7083 U/mL). Keywords: medicinal mushrooms, Pleurotus ostreatus, Ganoderma lucidum, total proteins, enzymes activities. Published in DKUM: 06.07.2022; Views: 1836; Downloads: 45
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8. Role of protein kinase network in excitation-contraction coupling in smooth muscle cellEtienne Roux, Prisca Mbikou, Aleš Fajmut, 2012, independent scientific component part or a chapter in a monograph Keywords: biofizika, encimi, gladke mišice, matematični modeli, biophysics, enzymes, smooth muscles, mathematical modelling Published in DKUM: 10.07.2015; Views: 1678; Downloads: 94
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9. Enzymatic fatty ester synthesisSimona Pečnik, Željko Knez, 1992, original scientific article Abstract: Fatty ester synthesis with immobilized 1,3-specific lipase from Mucor Miehei is described. 1,2-Isopropylidene glycerol was peoduced by condensation of glycerol with acetone was esterified with oleic acid in the presence of a Mucor Miehei lipaze (Lipozyme TM) to obtain 1,2 isopropylidene- 3-oleoyl glycerol. The effects of various process parameters (temperature and pressure)and various ratios (enzyme/substrate) have been investigated to determine optimal conditions for the esterification process. The highest conversion of oleic acid (80% w/w) was obtained at 55 oC and 57.057 bar, while the optimal addition of lipase to substrate was determined to be 0,096 g per gram of reaction mixture. The esterification can be modelled successfully as a reverse second order reaction. Thermodynamic properties of the reaction system at 55 oC and 0.057 bar were also determined. Activation energy was 20.82 kJ/mole, entropy of activation -0,26 kJ/(Kmole) and free energy of activation was 103.32 kJ/mole. Keywords: chemical engineering, biotechnology, esterification, syntheses, esters, enzymes, lipase, Mucor miehei, reaction kinetics, reaction thermodynamics, 1, 2-isopropylidene-3-oleoyl glycerol Published in DKUM: 06.06.2012; Views: 2549; Downloads: 100
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10. Silica aerogels as support for lipase catalyzed esterifications at sub- and supercritical conditionsZoran Novak, Maja Leitgeb, Vlasta Krmelj, Željko Knez, 2003, original scientific article Abstract: The enzymes (lipases from Candida rugosa and porcine pancreas) were immobilized on silica aerogels by sol-gel procedure followed by supercritical drying with CO2. Such immobilized enzymes were used as biocatalysts for esterification in supercritical CO2 and near critical propane at 40 °C and 100 bar. It was found out that the initial reaction rates in propane rose two to three times in comparison with the same reaction, catalyzed by free lipase. SC CO2 deactivated the non-immobilized lipase in reaction mixture while with the immobilized enzyme the conversion was 35%. The initial reaction rates in propane were 20 times higher than in water medium due to the properties of propane as a medium for esterification of fatty acids. Keywords: chemical processing, biotechnology, esterification, immobilization of enzymes, supercritial CO2, propane, lipases, supercritical CO2 drying Published in DKUM: 01.06.2012; Views: 1880; Downloads: 30
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