Application of molecular imprinting polymers in separation of active compounds from plants.

Molecular imprinting technique is becoming an appealing and prominent strategy to synthesize materials for target recognition and rapid separation. In recent years, it has been applied in separation of active compounds from various plants and has achieved satisfying results. This review aims to make a brief introduction of molecular imprinting polymers and their efficient application in the separation of various active components from plants, including flavonoids, organic acids, alkaloids, phenylpropanoids, anthraquinones, phenolics, terpenes, steroids, and diketones, which will provide some clues to help stimulating research into this fascinating and useful area.

Research Progress on Natural Diterpenoids in Reversing Multidrug Resistance.

Multidrug resistance (MDR) is one of the main impediments in successful chemotherapy in cancer treatment. Overexpression of ATP-binding cassette (ABC) transporter proteins is one of the most important mechanisms of MDR. Natural products have their unique advantages in reversing MDR, among which diterpenoids have attracted great attention of the researchers around the world. This review article summarizes and discusses the research progress on diterpenoids in reversing MDR.

Effect of Phlorotannins from Brown Algae Costaria costata on α-N-Acetylgalactosaminidase Produced by Duodenal Adenocarcinoma and Melanoma Cells.

The inhibitor of human α-N-acetylgalactosaminidase (α-NaGalase) was isolated from a water-ethanol extract of the brown algae Costaria costata. Currently, tumor α-NaGalase is considered to be a therapeutic target in the treatment of cancer. According to NMR spectroscopy and mass spectrometric analysis, it is a high-molecular-weight fraction of phlorethols with a degree of polymerization (DP) equaling 11-23 phloroglucinols (CcPh). It was shown that CcPh is a direct inhibitor of α-NaGalases isolated from HuTu 80 and SK-MEL-28 cells (IC50 0.14 ± 0.008 and 0.12 ± 0.004 mg/mL, respectively) and reduces the activity of this enzyme in HuTu 80 and SK-MEL-28 cells up to 50% at concentrations of 15.2 ± 9.5 and 5.7 ± 1.6 µg/mL, respectively. Molecular docking of the putative DP-15 oligophlorethol (P15OPh) and heptaphlorethol (PHPh) with human α-NaGalase (PDB ID 4DO4) showed that this compound forms a complex and interacts directly with the Asp 156 and Asp 217 catalytic residues of the enzyme in question. Thus, brown algae phlorethol CcPh is an effective marine-based natural inhibitor of the α-NaGalase of cancer cells and, therefore, has high therapeutic potential.

Deep-Sea Anemones Are Prospective Source of New Antimicrobial and Cytotoxic Compounds.

The peculiarities of the survival and adaptation of deep-sea organisms raise interest in the study of their metabolites as promising drugs. In this work, the hemolytic, cytotoxic, antimicrobial, and enzyme-inhibitory activities of tentacle extracts from five species of sea anemones (Cnidaria, orders Actiniaria and Corallimorpharia) collected near the Kuril and Commander Islands of the Far East of Russia were evaluated for the first time. The extracts of Liponema brevicorne and Actinostola callosa demonstrated maximal hemolytic activity, while high cytotoxic activity against murine splenocytes and Ehrlich carcinoma cells was found in the extract of Actinostola faeculenta. The extracts of Corallimorphus cf. pilatus demonstrated the greatest activity against Ehrlich carcinoma cells but were not toxic to mouse spleen cells. Sea anemones C. cf. pilatus and Stomphia coccinea are promising sources of antimicrobial and antifungal compounds, being active against Gram-positive bacteria Bacillus subtilis, Staphylococcus aureus, and yeast Candida albicans. Moreover, all sea anemones contain α-galactosidase inhibitors. Peptide mass fingerprinting of L. brevicorne and C. cf. pilatus extracts provided a wide range of peptides, predominantly with molecular masses of 4000-5900 Da, which may belong to a known or new structural class of toxins. The obtained data allow concluding that deep-sea anemones are a promising source of compounds for drug discovery.

Effects of Sponge-Derived Alkaloids on Activities of the Bacterial α-D-Galactosidase and Human Cancer Cell α-N-Acetylgalactosaminidase.

During a search for glycosidase inhibitors among marine natural products, we applied an integrated in vitro and in silico approach to evaluate the potency of some aaptamines and makaluvamines isolated from marine sponges on the hydrolyzing activity of α-N-acetylgalactosaminidase (α-NaGalase) from human cancer cells and the recombinant α-D-galactosidase (α-PsGal) from a marine bacterium Pseudoalteromonas sp. KMM 701. These alkaloids showed no direct inhibitory effect on the cancer α-NaGalase; but isoaaptamine (2), 9-demethylaaptamine (3), damirone B (6), and makaluvamine H (7) reduced the expression of the enzyme in the human colorectal adenocarcinoma cell line DLD-1 at 5 µM. Isoaaptamine (2), 9-demethylaaptamine (3), makaluvamine G (6), and zyzzyanone A (7) are slow-binding irreversible inhibitors of the bacterial α-PsGal with the inactivation rate constants (kinact) 0.12 min-1, 0.092 min-1, 0.079 min-1, and 0.037 min-1, as well as equilibrium inhibition constants (Ki) 2.70 µM, 300 µM, 411 µM, and 105 µM, respectively. Docking analysis revealed that these alkaloids bind in a pocket close to the catalytic amino acid residues Asp451 and Asp516 and form complexes, due to π-π interactions with the Trp308 residue and hydrogen bonds with the Lys449 residue. None of the studied alkaloids formed complexes with the active site of the human α-NaGalase.

Genomic Features of a Food-Derived Pseudomonas aeruginosa Strain PAEM and Biofilm-Associated Gene Expression under a Marine Bacterial α-Galactosidase.

The biofilm-producing strains of P. aeruginosa colonize various surfaces, including food products and industry equipment that can cause serious human and animal health problems. The biofilms enable microorganisms to evolve the resistance to antibiotics and disinfectants. Analysis of the P. aeruginosa strain (serotype O6, sequence type 2502), isolated from an environment of meat processing (PAEM) during a ready-to-cook product storage (-20 °C), showed both the mosaic similarity and differences between free-living and clinical strains by their coding DNA sequences. Therefore, a cold shock protein (CspA) has been suggested for consideration of the evolution probability of the cold-adapted P. aeruginosa strains. In addition, the study of the action of cold-active enzymes from marine bacteria against the food-derived pathogen could contribute to the methods for controlling P. aeruginosa biofilms. The genes responsible for bacterial biofilm regulation are predominantly controlled by quorum sensing, and they directly or indirectly participate in the synthesis of extracellular polysaccharides, which are the main element of the intercellular matrix. The levels of expression for 14 biofilm-associated genes of the food-derived P. aeruginosa strain PAEM in the presence of different concentrations of the glycoside hydrolase of family 36, α-galactosidase α-PsGal, from the marine bacterium Pseudoalteromonas sp. KMM 701 were determined. The real-time PCR data clustered these genes into five groups according to the pattern of positive or negative regulation of their expression in response to the action of α-galactosidase. The results revealed a dose-dependent mechanism of the enzymatic effect on the PAEM biofilm synthesis and dispersal genes.

Sponge-derived polybrominated diphenyl ethers and dibenzo-p-dioxins, irreversible inhibitors of the bacterial α-d-galactosidase.

An integrated in vitro and in silico approach was applied to evaluate the potency of hydroxylated polybrominated diphenyl ethers (OH-PBDEs) and spongiadioxins (OH-PBDDs) isolated from Dysidea sponges on the activity of the recombinant α-d-galactosidase of the GH36 family. It was revealed for the first time that all compounds rapidly and apparently irreversibly inhibited the bacterial α-d-galactosidase. The structure-activity relationship study in the series of OH-PBDEs showed that the presence of an additional hydroxyl group in 5 significantly enhanced the potency (IC50 4.26 µM); the increase of bromination in compounds from 1 to 3 increased their potency (IC50 41.8, 36.0, and 16.0 µM, respectively); the presence of a methoxy group decreased the potency (4, IC50 60.5 µM). Spongiadioxins 6, 7, and 8 (IC50 16.6, 33.1, and 28.6 µM, respectively) exhibited inhibitory action comparable to that of monohydroxylated diphenyl ethers 1-3. Docking analysis revealed that all compounds bind in a pocket close to the catalytic amino acid residues. Molecular docking detected significant compound-enzyme interactions in the binding sites of α-d-galactosidase. Superimposition of the enzyme-substrate and the enzyme-inhibitor complexes showed that their binding sites overlap.

Effect of Pentacyclic Guanidine Alkaloids from the Sponge Monanchora pulchra on Activity of α-Glycosidases from Marine Bacteria.

The effect of monanchomycalin B, monanhocicidin A, and normonanhocidin A isolated from the Northwest Pacific sample of the sponge Monanchora pulchra was investigated on the activity of α-galactosidase from the marine γ-proteobacterium Pseudoalteromonas sp. KMM 701 (α-PsGal), and α-N-acetylgalactosaminidase from the marine bacterium Arenibacter latericius KMM 426T (α-NaGa). All compounds are slow-binding irreversible inhibitors of α-PsGal, but have no effect on α-NaGa. A competitive inhibitor d-galactose protects α-PsGal against the inactivation. The inactivation rate (kinact) and equilibrium inhibition (Ki) constants of monanchomycalin B, monanchocidin A, and normonanchocidin A were 0.166 ± 0.029 min-1 and 7.70 ± 0.62 µM, 0.08 ± 0.003 min-1 and 15.08 ± 1.60 µM, 0.026 ± 0.000 min-1, and 4.15 ± 0.01 µM, respectively. The 2D-diagrams of α-PsGal complexes with the guanidine alkaloids were constructed with "vessel" and "anchor" parts of the compounds. Two alkaloid binding sites on the molecule of α-PsGal are shown. Carboxyl groups of the catalytic residues Asp451 and Asp516 of the α-PsGal active site interact with amino groups of "anchor" parts of the guanidine alkaloid molecules.

Development of host strains and vector system for an efficient genetic transformation of filamentous fungi.

An ability to synthesize extracellular enzymes degrading a wide spectrum of plant and algae polymeric substrates makes many fungi relevant for biotechnology. The terrestrial thermophilic and marine fungal isolates capable of plant and algae degradation have been tested for antibiotic resistance for their possible use in a new genetic transformation system. Plasmids encoding the hygromycin B phosphotransferase (hph) under the control of the cauliflower mosaic virus 35S promoter, the trpC gene promoter of Aspergillus nidulans, and the Aureobasidium pullulans TEF gene promoter were delivered into the fungal cells by electroporation. The effectiveness of different promoters was compared by transformation and growth of Thermothelomyces thermophila (formerly Myceliophthora thermophila) on the selective medium and by real-time PCR analysis. A highly efficient transformation was observed at an electric-pulse of 8.5 kV/cm by using 10 µg of DNA per 1 × 105 conidia. Although all promoters were capable of hph expression in the Th. thermophila cells, the trpC promoter provided the highest level of hygromycin resistance. We further successfully applied plant binary vector pPZP for co-transformation of hph gene and enhanced green fluorescent protein gene that confirmed this transformation system could be used as an appropriate tool for gene function studies and the expression of heterologous proteins in micromycetes.

Characterization of Properties and Transglycosylation Abilities of Recombinant α-Galactosidase from Cold-Adapted Marine Bacterium Pseudoalteromonas KMM 701 and Its C494N and D451A Mutants.

A novel wild-type recombinant cold-active α-d-galactosidase (α-PsGal) from the cold-adapted marine bacterium Pseudoalteromonas sp. KMM 701, and its mutants D451A and C494N, were studied in terms of their structural, physicochemical, and catalytic properties. Homology models of the three-dimensional α-PsGal structure, its active center, and complexes with D-galactose were constructed for identification of functionally important amino acid residues in the active site of the enzyme, using the crystal structure of the α-galactosidase from Lactobacillus acidophilus as a template. The circular dichroism spectra of the wild α-PsGal and mutant C494N were approximately identical. The C494N mutation decreased the efficiency of retaining the affinity of the enzyme to standard p-nitrophenyl-α-galactopiranoside (pNP-α-Gal). Thin-layer chromatography, matrix-assisted laser desorption/ionization mass spectrometry, and nuclear magnetic resonance spectroscopy methods were used to identify transglycosylation products in reaction mixtures. α-PsGal possessed a narrow acceptor specificity. Fructose, xylose, fucose, and glucose were inactive as acceptors in the transglycosylation reaction. α-PsGal synthesized -α(1→6)- and -α(1→4)-linked galactobiosides from melibiose as well as -α(1→6)- and -α(1→3)-linked p-nitrophenyl-digalactosides (Gal2-pNP) from pNP-α-Gal. The D451A mutation in the active center completely inactivated the enzyme. However, the substitution of C494N discontinued the Gal-α(1→3)-Gal-pNP synthesis and increased the Gal-α(1→4)-Gal yield compared to Gal-α(1→6)-Gal-pNP.

The Effect of Fucoidan from the Brown Alga Fucus evanescence on the Activity of α-N-Acetylgalactosaminidase of Human Colon Carcinoma Cells.

α-N-acetylgalactosaminidase (EC 3.2.1.49) (alpha-NaGalase) catalyzes the hydrolysis of N-acetamido-2-deoxy-α-d-galactoside residues from non-reducing ends of various complex carbohydrates and glycoconjugates. It is known that human cancer cells express an alpha-NaGalase, which accumulates in the blood plasma of patients. The enzyme deglycosylates the Gc protein-derived macrophage activating factor (GcMAF) and inhibits macrophage activity acting as an immunosuppressor. The high specific activity 0.033 ± 0.002 μmol mg−1 min−1 of the enzyme was found in human colon carcinoma cells DLD-1. The alpha-NaGalase of DLD-1 cells was isolated and biochemical characterized. The enzyme exhibits maximum activity at pH 5.2 and temperature 55 °C. The Km is 2.15 mM, Vmax⁻0.021 μmol min−1 mL−1, kcat⁻1.55 min−1 and kcat/Km⁻0.72 min−1 mM−1 at 37 °C, pH 5.2. The effects of fucoidan from the brown alga Fucus evanescence on the activity of alpha-NaGalase in human colon carcinoma DLD-1 cells and on the biosynthesis of this enzyme were investigated. It was shown that fucoidan did not inhibit free alpha-NaGalase, however, it reduced the expression of the enzyme in the DLD-1 cells at IC50 73 ± 4 μg mL−1.

Polaribacter staleyi sp. nov., a polysaccharide-degrading marine bacterium isolated from the red alga Ahnfeltia tobuchiensis.

A Gram-stain-negative, rod-shaped, motile by gliding and yellow-pigmented bacterium, designated strain 10Alg 139T, was isolated from the Pacific red alga Ahnfeltiato buchiensis. The phylogenetic analysis based on 16S rRNA gene sequences showed that the novel strain belonged to the genus Polaribacter, a member of the family Flavobacteriaceae, the phylum Bacteroidetes, with highest sequence similarity to Polaribacter butkevichii KMM 3938T (99.3 %) and 93.3-98.6 % to other recognized Polaribacter species. The prevalent fatty acids of strain 10Alg 139T were iso-C15 : 0 3-OH, C15 : 0 3-OH, iso-C15:0, iso-C13 : 0, C15 : 0 and C15 : 1ω6c. The polar lipid profile consisted of the major lipids phosphatidylethanolamine, two unidentified aminolipids and four unidentified lipids. The main respiratory quinone was menaquinone 6. The DNA G+C content of the type strain is 31.8 mol%. The new isolate and the type strains of recognized species of the genus Polaribacter were readily distinguished based on a number of phenotypic characteristics. A combination of the genotypic and phenotypic data showed that the isolate from alga represents a novel species of the genus Polaribacter, for which the name Polaribacterstaleyi sp. nov. is proposed. The type strain is 10Alg 139T (=KCTC 52773T=KMM 6729T).

Hooked on α-d-galactosidases: from biomedicine to enzymatic synthesis.

α-d-Galactosidases (EC 3.2.1.22) are enzymes employed in a number of useful bio-based applications. We have depicted a comprehensive general survey of α-d-galactosidases from different origin with special emphasis on marine example(s). The structures of natural α-galactosyl containing compounds are described. In addition to 3D structures and mechanisms of action of α-d-galactosidases, different sources, natural function and genetic regulation are also covered. Finally, hydrolytic and synthetic exploitations as free or immobilized biocatalysts are reviewed. Interest in the synthetic aspects during the next years is anticipated for access to important small molecules by green technology with an emphasis on alternative selectivity of this class of enzymes from different sources.

Stereochemical course of hydrolytic reaction catalyzed by alpha-galactosidase from cold adaptable marine bacterium of genus Pseudoalteromonas.

The recombinant α-galactosidase of the marine bacterium (α-PsGal) was synthesized with the use of the plasmid 40Gal, consisting of plasmid pET-40b (+) (Novagen) and the gene corresponding to the open reading frame of the mature α-galactosidase of marine bacterium Pseudoalteromonas sp. KMM 701, transformed into the Escherichia coli Rosetta(DE3) cells. In order to understand the mechanism of action, the stereochemistry of hydrolysis of 4-nitrophenyl α-D-galactopyranoside (4-NPGP) by α-PsGal was measured by (1)H NMR spectroscopy. The kinetics of formation of α- and ß-anomer of galactose showed that α-anomer initially formed and accumulated, and then an appreciable amount of ß-anomer appeared as a result of mutarotation. The data clearly show that the enzymatic hydrolysis of 4-NPGP proceeds with the retention of anomeric configuration, probably, due to a double displacement mechanism of reaction.

Flavobacterium ahnfeltiae sp. nov., a new marine polysaccharide-degrading bacterium isolated from a Pacific red alga.

A Gram-negative, aerobic, rod-shaped, motile by gliding and yellow-pigmented bacterium, designated strain 10Alg 130(T), that displayed the ability to destroy polysaccharides of red and brown algae, was isolated from the red alga Ahnfeltia tobuchiensis. The phylogenetic analysis based on 16S rRNA gene sequence placed the novel strain within the genus Flavobacterium, the type genus of the family Flavobacteriaceae, the phylum Bacteroidetes, with sequence similarities of 96.2 and 95.7 % to Flavobacterium jumunjiense KCTC 23618(T) and Flavobacterium ponti CCUG 58402(T), and 95.3-92.5 % to other recognized Flavobacterium species. The prevalent fatty acids of strain 10Alg 130(T) were iso-C15:0, iso-C15:0 3-OH, iso-C17:0 3-OH, C15:0 and iso-C17:1ω9c. The polar lipid profile consisted of phosphatidylethanolamine, two unknown aminolipids and three unknown lipids. The DNA G+C content of the type strain was 34.3 mol%. The new isolate and the type strains of recognized species of the genus Flavobacterium could strongly be distinguished by a number of phenotypic characteristics. A combination of the genotypic and phenotypic data showed that the algal isolate represents a novel species of the genus Flavobacterium, for which the name Flavobacterium ahnfeltiae sp. nov. is proposed. The type strain is 10Alg 130(T) (=KCTC 32467(T) = KMM 6686(T)).

Comparative analysis of glycoside hydrolases activities from phylogenetically diverse marine bacteria of the genus Arenibacter.

A total of 16 marine strains belonging to the genus Arenibacter, recovered from diverse microbial communities associated with various marine habitats and collected from different locations, were evaluated in degradation of natural polysaccharides and chromogenic glycosides. Most strains were affiliated with five recognized species, and some presented three new species within the genus Arenibacter. No strains contained enzymes depolymerizing polysaccharides, but synthesized a wide spectrum of glycosidases. Highly active ß-N-acetylglucosaminidases and α-N-acetylgalactosaminidases were the main glycosidases for all Arenibacter. The genes, encoding two new members of glycoside hydrolyses (GH) families, 20 and 109, were isolated and characterized from the genomes of Arenibacter latericius. Molecular genetic analysis using glycosidase-specific primers shows the absence of GH27 and GH36 genes. A sequence comparison with functionally-characterized GH20 and GH109 enzymes shows that both sequences are closest to the enzymes of chitinolytic bacteria Vibrio furnissii and Cellulomonas fimi of marine and terrestrial origin, as well as human pathogen Elisabethkingia meningoseptica and simbionts Akkermansia muciniphila, gut and non-gut Bacteroides, respectively. These results revealed that the genus Arenibacter is a highly taxonomic diverse group of microorganisms, which can participate in degradation of natural polymers in marine environments depending on their niche and habitat adaptations. They are new prospective candidates for biotechnological applications due to their production of unique glycosidases.

Molecular characterization and therapeutic potential of a marine bacterium Pseudoalteromonas sp. KMM 701 alpha-galactosidase.

An alpha-galactosidase capable of converting B red blood cells into the universal blood type cells at the neutral pH was produced by a novel obligate marine bacterium strain KMM 701 (VKM B-2135 D). The organism is heterotrophic, aerobic, and halophilic and requires Na+ ions and temperature up to 34 degrees C for its growth. The strain has a unique combination of polysaccharide-degrading enzymes. Its single intracellular alpha-galactosidase exceeded other glycoside hydrolases in the level of expression up to 20-fold. The alpha-galactosidase was purified to determine the N-terminal amino acid sequences and new activities. It was found to inhibit Corynebacterium diphtheria adhesion to host buccal epithelium cell surfaces with high effectiveness. The nucleotide sequence of the homodimeric alpha-galactosidase indicates that its subunit is composed of 710 amino acid residues with a calculated Mr of 80,055. This alpha-galactosidase shares structural property with 36 family glycoside hydrolases. The properties of the enzyme are likely to be highly beneficial for medicinal purposes.

An endo-(1-->3)-beta-D-glucanase from the scallop Chlamys albidus: catalytic properties, cDNA cloning and secondary-structure characterization.

An endo-(1-->3)-beta-d-glucanase (L(0)) with molecular mass of 37 kDa was purified to homogeneity from the crystalline style of the scallop Chlamys albidus. The endo-(1-->3)-beta-d-glucanase was extremely thermolabile with a half-life of 10 min at 37 degrees C. L(0) hydrolyzed laminaran with K(m) approximately 0.75 mg/mL, and catalyzed effectively transglycosylation reactions with laminaran as donor and p-nitrophenyl betad-glucoside as acceptor (K(m) approximately 2mg/mL for laminaran) and laminaran as donor and as acceptor (K(m) approximately 5mg/mL) yielding p-nitrophenyl betad-glucooligosaccharides (n=2-6) and high-molecular branching (1-->3),(1-->6)-beta-d-glucans, respectively. Efficiency of hydrolysis and transglycosylation processes depended on the substrate structure and decreased appreciably with the increase of the percentage of beta-(1-->6)-glycosidic bonds, and laminaran with 10% of beta-(1-->6)-glycosidic bonds was the optimal substrate for both reactions. The CD spectrum of L(0) was characteristic for a protein with prevailing beta secondary-structural elements. Binding L(0) with d-glucose as the best acceptor for transglycosylation was investigated by the methods of intrinsic tryptophan fluorescence and CD. Glucose in concentration sufficient to saturate the enzyme binding sites resulted in a red shift in the maximum of fluorescence emission of 1-1.5 nm and quenching the Trp fluorescence up to 50%. An apparent association constant of L(0) with glucose (K(a)=7.4 x 10(5)+/-1.1 x 10(5)M(-1)) and stoichiometry (n=13.3+/-0.7) was calculated. The cDNA encoding L(0) was sequenced, and the enzyme was classified in glycoside hydrolases family 16 on the basis of the amino acid sequence similarity.

Structure, biological activity, and enzymatic transformation of fucoidans from the brown seaweeds.

Recent advances in the study of fucoidans, biologically active sulfated alpha-L-fucans of diverse structures and synthesized exclusively by marine organisms, are overviewed. Their structure, biological activity, the products of their enzymatic degradation and the different enzymes of degradation and modification are considered.

A new precursor for the immobilization of enzymes inside sol-gel-derived hybrid silica nanocomposites containing polysaccharides.

Tetrakis(2-hydroxyethyl) orthosilicate (THEOS) introduced by Hoffmann et al. (J. Phys. Chem. B., 106 (2002) 1528) was first used to prepare hybrid nanocomposites containing various polysaccharides and immobilize enzymes in these materials. Two different types of O-glycoside hydrolyses (EC3.2.1), 1-->3-beta-D-glucanase LIV from marine mollusk Spisula sacchalinensis and alpha-D-galactosidase from marine bacterium Pseudoalteromonas sp. KMM 701, were taken for the immobilization. To reveal whether the polysaccharide inside the hybrid material influences the enzyme entrapment and functioning, negatively charged xanthan, cationic derivative of hydroxyethylcellulose and uncharged locust bean gum were examined. The mechanical properties of these nanocomposites were characterized by a dynamic rheology and their structure by a scanning electron microscopy. It was found that 1-->3-beta-D-glucanase was usually immobilized without the loss of its activity, while the alpha-D-galactosidase activity in the immobilized state depended on the polysaccharide type of material. An important point is that the amount of immobilized enzymes was small, comparable to their content in the living cells. It was shown by the scanning electron microscopy that the hybrid nanocomposites are sufficiently porous that allows the enzymatic substrates and products to diffuse from an external aqueous solution to the enzymes, whereas protein molecules were immobilized firmly and not easily washed out of the silica matrix. A sharp increase of the enzyme lifetime (more than a hundred times) was observed after the immobilization. As established, the efficient entrapment of enzymes is caused by few advantages of new precursor over the currently used TEOS and TMOS: (i) organic solvents and catalysts are not needed owing to the complete solubility of THEOS in water and the catalytic effect of polysaccharides on the sol-gel processes; (ii) the entrapment of enzymes can be performed at any pH which is suitable for their structural integrity and functionality; (iii) a gel can be prepared at reduced concentrations of THEOS (1-2%) in the initial solution that excludes a notable heat release in the course of its hydrolysis.