1. Influence of supercritical carbon dioxide on the activity and conformational changes of α-amylase, lipase, and peroxidase in the solid state using white wheat flour as an exampleMilena Ivanović, Željko Knez, Maja Leitgeb, 2023, original scientific article Keywords: enzyme inactivation, supercritical carbon dioxide, a-amilaze, lipase, antioxidants, horseradish peroxidase, structural changes, CD spectroscopy
Published in DKUM: 22.04.2024; Views: 183; Downloads: 8
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2. Monocarbonyl Curcumin Analogues as Potent Inhibitors against Human Glutathione Transferase P1-1Panagiota D. Pantiora, Veronika Furlan, Dimitris Matiadis, Barbara Mavroidi, Fereniki Perperopoulou, Anastassios C. Papageorgiou, Marina Sagnou, Urban Bren, Maria Pelecanou, Nikolaos E. Labrou, 2023, original scientific article Abstract: The isoenzyme of human glutathione transferase P1-1 (hGSTP1-1) is involved in multi-drug resistance (MDR) mechanisms in numerous cancer cell lines. In the present study, the inhibition potency of two curcuminoids and eleven monocarbonyl curcumin analogues against hGSTP1-1 was investigated. Demethoxycurcumin (Curcumin II) and three of the monocarbonyl curcumin analogues exhibited the highest inhibitory activity towards hGSTP1-1 with IC50 values ranging between 5.45 1.08 and 37.72 1.02 M. Kinetic inhibition studies of the most potent inhibitors demonstrated that they function as non-competitive/mixed-type inhibitors. These compounds were also evaluated for their toxicity against the prostate cancer cells DU-145. Interestingly, the strongest hGSTP1-1 inhibitor, (DM96), exhibited the highest cytotoxicity with an IC50 of 8.60 1.07 M, while the IC50 values of the rest of the compounds ranged between 44.59–48.52 M. Structural analysis employing molecular docking, molecular dynamics (MD) simulations, and binding-free-energy calculations was performed to study the four most potent curcumin analogues as hGSTP1-1 inhibitors. According to the obtained computational results, DM96 exhibited the lowest binding free energy, which is in agreement with the experimental data. All studied curcumin analogues were found to form hydrophobic interactions with the residue Gln52, as well as hydrogen bonds with the nearby residues Gln65 and Asn67. Additional hydrophobic interactions with the residues Phe9 and Val36 as well as – stacking interaction with Phe9 contributed to the superior inhibitory activity of DM96. The van derWaals component through shape complementarity was found to play the most important role in DM96-inhibitory activity. Overall, our results revealed that the monocarbonyl curcumin derivative DM96 acts as a strong hGSTP1-1 inhibitor, exerts high prostate cancer cell cytotoxicity, and may, therefore, be exploited for the suppression and chemosensitization of cancer cells. This study provides new insights into the development of safe and effective GST-targeted cancer chemosensitizers.
Keywords: curcuminoids, curcumin analogues, human glutathione transferase P1-1 (hGSTP1-1), glutathione transferase, enzyme inhibition, multi-drug resistance
Published in DKUM: 12.03.2024; Views: 288; Downloads: 13
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3. Transglutaminase in foods and biotechnologyKatja Vasić, Željko Knez, Maja Leitgeb, 2023, review article Keywords: transglutaminase, industrial enzyme, protein-modifying enzyme, crosslinker, antibody– drug conjugation, immobilization
Published in DKUM: 06.12.2023; Views: 563; Downloads: 22
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5. Use of non-conventional cell disruption method for extraction of proteins from black yeastsMaja Čolnik, Mateja Primožič, Željko Knez, Maja Leitgeb, 2016, original scientific article Abstract: The influence of pressure and treatment time on cells disruption of different black yeasts and on activities of extracted proteins using supercritical carbon dioxide process was studied. The cells of three different black yeasts Phaeotheca triangularis, Trimatostroma salinum, and Wallemia ichthyophaga were exposed to supercritical carbon dioxide (SC $CO_2$) by varying pressure at fixed temperature (35°C). The black yeasts cell walls were disrupted, and the content of the cells was spilled into the liquid medium. The impact of SC $CO_2$ conditions on secretion of enzymes and proteins from black yeast cells suspension was studied. The residual activity of the enzymes cellulase, $beta$-glucosidase, $alpha$-amylase, and protease was studied by enzymatic assay. The viability of black yeast cells was determined by measuring the optical density of the cell suspension at 600 nm. The total protein concentration in the suspension was determined on UV–Vis spectrophotometer at 595 nm. The release of intracellular and extracellular products from black yeast cells was achieved. Also, the observation by an environmental scanning electron microscopy shows major morphological changes with SC $CO_2$-treated cells. The advantages of the proposed method are in a simple use, which is also possible for heat-sensitive materials on one hand and on the other hand integration of the extraction of enzymes and their use in biocatalytical reactions.
Keywords: P. triangularis, W. ichtyophaga, T. salinum, supercritical carbon dioxide, enzyme activity, cells
Published in DKUM: 10.07.2017; Views: 1546; Downloads: 390
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6. Pressure stability of lipases and their use in different systemsMaja Leitgeb, Željko Knez, 2001, original scientific article Abstract: For the investigation of the solvent impact on the enzymes, lipases from different sources (Pseudomonas fluorescences, Rhizopus javanicus, Rhizopus niveus, Candida rugose and Porcine pancreas) were used. Stability and activity of these lipases in aqueous medium in supercritical $CO_2$ and liquid propane at 100 bar and 40°C were studied.
On the basis of previous results lipases were used for their application in two different systems. The application of the polysulphone membrane in the continuous stirred tank membrane reactor was studied on the model system of the hydrolysis of oleyl oleate in propane at high pressure. As a catalyst the Candida rugosa lipase was used. The next utilization of lipases was the use of on silica arerogel self-immobilized lipase from Porcine pancreas as catalyst for esterification reaction in near-critical propane at 40°C and 100 bar.
Keywords: chemical processing, supercritical fluids, lipases, enzyme stability, high pressure membrane reactor
Published in DKUM: 10.07.2015; Views: 1541; Downloads: 196
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7. MLC-kinase/phosphatase control of Ca[sup]2+ signal transduction in airway smooth musclesAleš Fajmut, Milan Brumen, 2008, original scientific article Abstract: In airway smooth muscles, kinase/phosphatase-dependent phosphorylation and dephosphorylation of the myosin light chain (MLC) have been revealed by many authors as important steps in calcium ▫$(Ca^{2+})$▫ signalling pathway from the variation of ▫$Ca^{2+}$▫ concentration in cytosol to the force development. Here, a theoretical analysis of the control action of MLC-kinase (MLCK) and MLC-phosphatase (MLCP) in ▫$Ca^{2+}$▫ signalling is presented and related to the general control principles of these enzymes, which were previously studied by Reinhart Heinrich and his co-workers. The kinetic scheme of the mathematical model considers interactions among ▫$Ca^{2+}$▫, calmodulin (CaM) and MLCK and the well-known 4-state actomyosin latch bridge model, whereby a link between them is accomplished by the conservation relation of all species of MLCK. The mathematical model predicts the magnitude and velocity of isometric force in smooth muscles upon transient biphasic ▫$Ca^{2+}$▫ signal. The properties of signal transduction in the system such as the signalling time, signal duration and signal amplitude, which are reflected in the properties of force developed, are studied by the principles of the metabolic control theory. The analysis of our model predictions confirms as shown by Reinhart Heinrich and his co-workers that MLCK controls the amplitude of signal more than its duration, whereas MLCP controls both. Finally, the simulations of elevated total content of MLCK, a typical feature of bronchial muscles of asthmatic subjects and spontaneously hypertensive rats as well as potentiation of MLCP catalytic activity, are carried out and are discussed in view of an increase in the force magnitude.
Keywords: cells, calcium, calcium oscillations, myosin light chains, enzyme activities, mathematical models
Published in DKUM: 07.06.2012; Views: 2348; Downloads: 33
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8. The influence of enzymatic treatment on wool fibre properties using PEG-modified proteasesSuzana Jus, Marc Schroeder, Georg M. Gübitz, Elisabeth Heine, Vanja Kokol, 2007, original scientific article Abstract: The main contribution of the presented work was to introduce the use of proteases modified with the soluble polymer polyethylene glycol (PEG) in the bio-finishing process of wool fibres, to target enzyme action to the outerparts of wool fibres, i.e. to avoid the diffusion and consequent destroying of the inner parts of the wool fibre structure, in the case of native proteases using. Different proteolytic enzymes from Bacillus lentus and Bacillus subtilis in native and PEG-modified forms were investigated and their influence on the modification of wool fibres morphology surface, chemical structure, as well as the hydrolysis of wool proteins, the physico-mechanical properties, and the sorption properties of 1:2 metal complex dye during dyeing were studied. SEM images of wool fibres confirmed smoother and cleaner fibre surfaces without fibre damages using PEG-modified proteases. Modified enzyme products have a benefit effect on the wool fibres felting behaviours (14%) in the case when PEG-modified B. lentus is used, without markedly fibre damage expressed by tensile strength and weight loss ofthe fibre. Meanwhile the dye exhaustion showed slower but comparable level of dye uptake at the end of the dyeing.
Keywords: volnena vlakna, proteolitski encimi, encimske modifikacije, sorpcija barve, morfologija vlaken, wool fibres, proteolytic enzymes, enzyme modification, felting, dye sorption, protein hydrolysis, XPS-analysis, fibre morphology
Published in DKUM: 01.06.2012; Views: 3130; Downloads: 109
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9. Enzymatic reactions in dense gasesŽeljko Knez, 2009, review article Abstract: The developments on applications of supercritical fluids as alternative solvents for biocatalytic processes that have taken place over the past two decades have been reviewed. An overview of process parameters influencing enzyme activity and stability, the influence of process parameters on reaction rates and productivity are presented. Applications of various types of reactors for enzymatic reaction in dense fluids, limitations of using enzymes as biocatalyst in supercritical fluids as well as future trends are presented. Main advantages of using dense gases as solvents for biocatalyzed reactions are the tunability of solvent properties and simple down stream processing features that can be readily combined with other unit operations. Although many enzymes are stable in supercritical fluids (SCFs) one should pay considerable attention to finding the correct reaction conditions for each substrate/enzyme/SCF system. One of the persistent problems is the instability and deactivation of enzymes under pressure and temperature. At present the most stable enzymes are hydrolases (lipases and esterases) for which pressure effect is lower than temperature deactivation.
Keywords: biocatalysis, supercritical fluids, enzyme bioreactors, heterogeneous biocatalysis
Published in DKUM: 01.06.2012; Views: 2577; Downloads: 99
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10. Stability of proteinase from Carica papaya latex in dense gasesMaja Leitgeb, Mateja Primožič, Željko Knez, 2005, original scientific article Abstract: Proteinase from Carica papaya latex was tested on its thermal stability at atmospheric pressure and in supercritical carbon dioxide, near-critical propane and dimethyl-ether. In supercritical carbon dioxide at 300 bar thermalactivation of the examined proteinase was improved in the comparison toatmospheric pressure. In propane and dimethyl-ether (300 bar) activity of the examined proteinase decreased. Influence of compressionžexpansion cycles on residual activity of the same proteinase in supercritical carbon dioxide (300 bar and 50 °C) was studied, as well. Different ways of transition from supercritical to low-pressure-state were used which affected residual activityof the proteinase.Addition of water in the system increased activity of proteinase from C. papaya, which was incubated in supercritical carbon dioxide for 24 h. Optimum amount of water was found to be between 0.5 and 0.7 g/L.
Keywords: chemical processing, high pressure technology, supercritical CO2, proteinase, thermal stability, pressure stability, enzyme activity, water content, dense gases
Published in DKUM: 01.06.2012; Views: 2015; Downloads: 28
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