Mechanisms of UV-induced human lymphocyte apoptosis.

The article reviews the results of the studies of marker parameters (indicators) of various pathways and mechanisms of apoptosis of lymphocytes in donor peripheral blood induced by UV light (240-390 nm) in doses of 151, 1510, and 3020 J/m2. The article analyses the processes of DNA fragmentation, distortion of the structural asymmetry of the cell membranes, changes in the degree of DNA damage (single-strand breaks), transcriptional factor р53, cytochrome с, Fas receptors (CD95), caspase-3, caspase-8, and caspase-9, reactive oxygen species, and calcium ions in UV modified cells. The study determined that programmed cell death of lymphocytes after UV irradiation with 1510 J/m2 involves the р53-dependent pathway of the nuclear mechanism, as well as receptor-mediated caspase mechanism, mitochondrial mechanism, and the mechanism associated with the defects in calcium homeostasis. Cell death is mediated by reactive oxygen and calcium ions. The article suggests a scheme of possible intracellular events resulting in the apoptotic death of lymphocytes after UV irradiation.

Novel biocatalysts based on enzymes in complexes with nano- and micromaterials.

In today's world, there is a wide array of materials engineered at the nano- and microscale, with numerous applications attributed to these innovations. This review aims to provide a concise overview of how nano- and micromaterials are utilized for enzyme immobilization. Enzymes act as eco-friendly biocatalysts extensively used in various industries and medicine. However, their widespread adoption faces challenges due to factors such as enzyme instability under different conditions, resulting in reduced effectiveness, high costs, and limited reusability. To address these issues, researchers have explored immobilization techniques using nano- and microscale materials as a potential solution. Such techniques offer the promise of enhancing enzyme stability against varying temperatures, solvents, pH levels, pollutants, and impurities. Consequently, enzyme immobilization remains a subject of great interest within both the scientific community and the industrial sector. As of now, the primary goal of enzyme immobilization is not solely limited to enabling reusability and stability. It has been demonstrated as a powerful tool to enhance various enzyme properties and improve biocatalyst performance and characteristics. The integration of nano- and microscale materials into biomedical devices is seamless, given the similarity in size to most biological systems. Common materials employed in developing these nanotechnology products include synthetic polymers, carbon-based nanomaterials, magnetic micro- and nanoparticles, metal and metal oxide nanoparticles, metal-organic frameworks, nano-sized mesoporous hydrogen-bonded organic frameworks, protein-based nano-delivery systems, lipid-based nano- and micromaterials, and polysaccharide-based nanoparticles.

Efficient fructose production from plant extracts by immobilized inulinases from Kluyveromyces marxianus and Helianthus tuberosus.

The enzymatic hydrolysis of poly- and oligosaccharides from plants seems like an advantageous approach for sugars production. Two inulinases producing fructose from plant oligosaccharides were isolated from yeast Kluyveromyces marxianus and plant Helianthus tuberosus. Both enzymes were immobilized on polymeric carriers by using the static adsorption approach. We could save 80.4% of the initial catalytic activity of plant inulinase immobilized on KU-2 cation-exchange resin and 75.5% of yeast enzyme activity adsorbed on AV-17-2P anion-exchange resin. After immobilization, the Km values increased 1.5 and 6 times for enzymes from K. marxianus and H. tuberosus, respectively. The optimal temperatures for catalysis of both enzymes were increased from 48-50 °C up to 70 °C. The activities of both immobilized enzymes remained unchanged after the 10 cycles of 20-min hydrolysis reaction at 70 °C model batch reactor. Sorbents, native and immobilized enzymes did not exhibit any mutagenic or cytotoxic activity.

[UV-Modification of Free and Immobilized Trypsin].

We investigated the mechanism of UV-radiation influence on trypsin in free and immobilized (on chitosan) states. The catalytic activity of free enzyme under the action of UV-light is subjected to changes to a greater extent than that of the immobilized one. We assume that the photoprotection effect of chitosan is caused for the following reasons: firstly, through interactaction with trypsin molecules chitosan forms a more photoresistant complex as compared to the native protein; secondly, chitosan probably binds the active photopro- ducts of a free radical nature, thus preventing oxidation (destruction) of several amino acids of the enzyme under its UV-radiation.

In silico design of high-affinity ligands for the immobilization of inulinase.

Using computer modeling, virtual screening of high-affinity ligands for immobilization of inulinase - an enzyme that cleaves inulin and fructose-containing polymers to fructose - has been performed. The inulinase molecule from Aspergillus ficuum (pdb: 3SC7) taken from the database of protein structures was used as a protein model and the target for flexible docking. The set of ligands studied included simple sugars (activators, inhibitors, products of enzymatic catalysis), as well as high-molecular weight compounds (polycation and polyanion exchange resins, glycoproteins, phenylalanine-proline peptide, polylactate, and caffeine). Based on the comparative analysis of the values of the total energy and the localization of ligand binding sites, we made several assumptions concerning the mechanisms of interaction of the suggested matrices for the immobilization of enzyme molecules and the structural features of such complexes. It was also assumed that the candidates for immobilization agents meeting the industrial requirements may be glycoproteins, for which we propose an additional incorporation of cysteine residues into their structure, aimed to create disulfide «anchors¼ to the surface.

[Research of UV-Induced Changes in the Structural and Functional Properties of Inulinases].

UV-induced changes in the catalytic activity and radiuses of inulinases molecules from various producers (plants, fungy, yeast) are studied. It is established that specific enzymes activity and the sizes of inulinases molecules from Helianthus tuberosus and Kluyveromyces marxianus under the influence of UV-light in the ranges of doses 4530-6040 and 755-6040 J/m2, respectively, are subjected to changes more than structural and functional characteristics of inulinase fromAspergillus niger. It is probably connected with lower contents in it of aromatic amino acids such as tyrosine and phenylalanine. The most expressed loss of functional properties of inulinase from Helianthus tuberosus can be caused by the'existence of significantly more numbers of cysteine in plant fructan-exohydrolases in relation to microbic enzymes. A scheme for the stages of response of inulinases of various origins on the influence of UV-light in a certain range of radiation doses is offered.

[Investigation of Inulinase Permolecular Organization from Producers of the Genus Aspergillus by Means of Some Computing and Experimental Methods].

The computer model for a dimer of inulinase from Aspergillus ficuum is designed. The permolecular organization of inulinase from Aspergillus niger is experimentally investigated. The question about the role of various inulinase forms in manifestation of their functional activity is discussed. It is shown, that in the process of inulinase dimerization when contacts between monomeric forms of the enzyme are formed, a key role belongs to the nonpolar amino acid residues.

[Investigation of mechanisms of interaction between inulinase from Kluyveromyces marxianus and the matrices of ion-exchange resins and fiber].

It is established that ion exchange resins AV-17-2P, KU-2, AV-16-GS, AM 21A, IMAC-HP, PUROLITE and fiber VION KN-1 can be applied as carriers for inulinase immobilization. The analysis of IR spectra for an enzyme, carriers and heterogeneous enzyme preparations showed that inulinase binding to matrices of various carriers occurs in general through electrostatic interactions. It is assumed that the mechanisms of interaction between inulinase from Kluyveromyces marxianus and the matrices of cation- and anion exchange polymers differ essentially from each other: different sites of protein molecule take part in adsorption that causes various conformational reorganizations in an enzyme molecule.

Inulinase immobilization on macroporous anion-exchange resins by different methods.

Inulinase was immobilized on AV-16-GS macroporous anion-exchange resin by physical and chemical methods. Binding of the enzyme to the carrier by the studied methods led to a shift of catalysis optimal temperature towards higher values and to extension of the range of optimum pH values. Our modification of glutaraldehyde method of inulinase immobilization increased catalytic activity of the preparation in comparison with the common glutaraldehyde method.

Evaluation of the number of ionogenic groups of inulinase by acid-base titration.

Acid base titration showed that Aspergillus awamori inulinase includes 178 asparaginic and glutamic acid residues, 20 histidine, 10 serine, and 34 lysine and tyrosine residues. Denaturation temperature for this enzyme was calculated using analysis of the proportion of stabilizing and destabilizing amino acids in the molecule.