M. G. Holyavka

Inulinases from various producers: The features of their permolecular organization

The structural organization of inulinases from yeasts, fungi, and plants are researched. For studying their sizes, molecular weight, and permolecular organization, an approach consisting of a combination of atomic force microscopy with methods of dynamic light scattering, gel chromatography, and electrophoresis was used. It is shown that inulinases from Kluyveromyces marxianus and Aspergillus niger form geterodimers and inulinases from tubers of Helianthus tuberosus are present as both dimers and monomers. The role of various forms in the functional activity of inulinase molecules is discussed.

Targeting microbial biofilms using Ficin, a nonspecific plant protease

Biofilms, the communities of surface-attached bacteria embedded into extracellular matrix, are ubiquitous microbial consortia securing the effective resistance of constituent cells to environmental impacts and host immune responses. Biofilm-embedded bacteria are generally inaccessible for antimicrobials, therefore the disruption of biofilm matrix is the potent approach to eradicate microbial biofilms. We demonstrate here the destruction of Staphylococcus aureus and Staphylococcus epidermidis biofilms with Ficin, a nonspecific plant protease. The biofilm thickness decreased two-fold after 24 hours treatment with Ficin at 10 μg/ml and six-fold at 1000 μg/ml concentration. We confirmed the successful destruction of biofilm structures and the significant decrease of non-specific bacterial adhesion to the surfaces after Ficin treatment using confocal laser scanning and atomic force microscopy. Importantly, Ficin treatment enhanced the effects of antibiotics on biofilms-embedded cells via disruption of biofilm matrices. Pre-treatment with Ficin (1000 μg/ml) considerably reduced the concentrations of ciprofloxacin and bezalkonium chloride required to suppress the viable Staphylococci by 3 orders of magnitude. We also demonstrated that Ficin is not cytotoxic towards human breast adenocarcinoma cells (MCF7) and dog adipose derived stem cells. Overall, Ficin is a potent tool for staphylococcal biofilm treatment and fabrication of novel antimicrobial therapeutics for medical and veterinary applications.

Structural and functional properties of inulinases: A review

Abstract The studies of structural and functional properties of inulinases and their molecular and supramolecular organization are crucial for understanding the functional mechanisms for key enzymes of polyfructans metabolism which demands special attention. This review addresses these issues with a focus to disagreement concerning supramolecular organization of inulinases, practical importance of different glycosylation degrees, and mechanism of splitting of glycosidic linkages, which occur in different organisms.

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 «anchors» to the surface.

Structural and functional properties of inulinases. Ways to regulate their activity

The presented review is devoted to the analysis of structural and functional properties of inulinases from various producers. A special attention is given to the questions of molecular and permolecular organization of enzymes, description of their functional features depending upon their structural conditions and in various microenvironment. Perspectives of the development of biotechnological processes with the use of free and immobilized inulinases are discussed from a biophysical viewpoint.

The molecular mechanism of adsorption immobilization of inulinase on polymer matrices

The conditions and mechanisms of the immobilization of inulinase on polymeric carriers were studied using the VION KN-1 and KU-2 cation-exchangers, VION AN-1 and AV-17-2P anion-exchangers, and the ampholyte KOPAN-90. The calculated data showed a significant role of van der Waals interactions and hydrogen bonding in the formation of virtually all inulinase complexes with the immobilization matrices. The AV-17-2P anion-exchanger was the only one of the studied polymer matrices that was unable to form hydrogen bonds with inulinase. The mechanisms of the interaction between inulinase and various ampholytes and cation and anion exchange resins differ from each other. The strongest differences are observed in mechanisms of the sorption of inulinase on VION KN-1 and chitosan matrices. Approximately 87% of the identical amino-acid residues are involved in the interaction of the enzyme with the KU-2 and AV-17-2P resins and the VION AN-1 and KOPAN-90 fibers.

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.

A Rapid Method for Secondary-Structure Analysis of the Inulinases of Different Microbial Producers

Computer simulation of secondary structures (calculation of the ratio of α-helices, β-sheets and disordered regions) is a perspective tool needed at the initial stages of the studies of structural and functional features of inulinases, since it enables one to estimate the fluctuation ranges of tested indicators. However, the data from computations should be verified by a number of biophysical and biochemical experimental data, in particular, by experiments using IR-spectroscopy. In the present work, the difference between the experimental and computational data was 3–4% for inulinase from Aspergillus awamori and 12–18% for the enzyme from Kluyveromyces marxianus. Consequently, the analysis of secondary structures of enzymes is applicable for making rapid predictions of the fluctuation ranges of physical−chemical and kinetic characteristics of protein molecules, as well as for rapid evaluation of their dynamic state.

The interaction of inulinase molecules with a chitosan matrix: UV-induced changes in the functional properties of immobilized inulinase

We have developed a technique for the immobilization of inulinase on chitosans with different molecular weights. The acid-soluble mid-molecular-weight (200 kDa) and high-molecular-weight (350 kDa) chitosans are shown to be promising matrices for inulinase adsorption. We assumed that the formation of an inulinase-chitosan matrix complex occurs mainly due to hydrophobic interactions; electrostatic interactions also play an essential role. The enzyme complex with high-molecular-weight chitosan is more stable against the action of UV light and temperature. This allows us to recommend chitosan as a catalyst in industry for the production of fructose from inulin-containing vegetable raw mateials.

Reconstruction of the spatial structure of inulinase from Kluyveromyces marxianus to find regulatory pathways of its catalytic activity

Reconstruction of the spatial structure of inulinase (EC 3.2.1.7) from Kluyveromyces marxianus (an enzyme that hydrolyzes inulin and other fructose-based polymers to fructose) was carried out by highthroughput computational modeling. A structural model of a closely related homologous protein, viz., invertase from yeast Saccharomyces cerevisiae (PDB-ID: 4EQV), was used as a template. The reconstructed model can be used for computer calculations for optimizing the biotechnological feasibility of inulinase.