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The aim of this work was the study of native and galactosidases from Penicillium canescens under denaturing conditions caused by guanidine hydrochloride. Calculation of kinetics and constants of enzymes inactivation was carried out on using experimental kinetic curves of enzyme denaturation. We observed significant differences in the kinetics of inactivation of native and deglycosylated α-galactosidases from P. canescens caused by guanidine hydrochloride. Native enzyme was stable within the selected range of guanidine hydrochloride concentrations (from 0.1 to 3.0 M), retaining no less than 50% of the initial enzyme activity for 3 days. Deglycosylated enzyme preparations were less stable and they lost their activity within 5–30 minutes, when they were treated with guanidine hydrochloride in concentrations above 1 M. Dissociation rate constant of native and deglycosylated forms of the enzyme differed by 10 to 100 folds. It was shown that subunit interactions play a major role in the process of inactivation of the enzyme, and the carbohydrate component is essential for stabilizing of subunit bonds and maintaining conformational stability of the enzyme under denaturing conditions of chemical agents.

One of the important problems of current biotechnology is the usage of enzymes of microbial origin for destruction of poorly soluble compounds and synthesis of new drugs. In recent years a great deal of researchers’ attention has been given to such technologically promising carbohydrases as O-glycosylhydrolases catalyzing the hydrolysis of O-glycoside links in glycosides, oligo- and polysaccharides, glycolipids, and other glycoconjugates. Aim. The review provides data on the position of α-L-rhamnosidases in the modern hierarchical classification of glycosidases and presents data available in the literature on the features of the enzyme structure in various microorganisms. Methods. The publications from the following databases were analyzed: PubMed (https://pubmed. nsbi.nlm.nih.gov/), the Carbohydrate-Active enZYmes (http://www.cazy.org/), the BRENDA Enzyme Database (https://www.brenda-enzymes.org/). Results. Data on the physicochemical, catalytic, and kinetic properties of α-L-rhamnosidases in microorganisms of different taxonomic groups have been systematized. The peculiarities of the substrate specificity of the enzyme depending on the nature of the protein and the growing conditions of the producer are characterized. Conclusions. Functional properties and specificity action of microbial α-L-rhamnosidases suggest their broad-range applicability for food and animal feed processing, as well as obtaining biologically active compounds for the pharmaceutical industry and medicine. [ABSTRACT FROM AUTHOR]

α-L-Rhamnosidase (α-L-rhamnoside-rhamnohydrolase EC 3.2.1.40) showing specificity for terminal α-1,2-, α-1,4- and α-1,6-linked rhamnose residues, which oft en present in glycoconjugates and synthetic glycosides, can be successfully used in biotechnology for the hydrolysis of rhamnopyranoside residues present in some bioflavonoids, glycoproteins, glycolipids, and other glycoconjugates. Previously, we have shown that a significant part of the coordination compounds of various metals act as effectors of the activity of α-L-rhamnosidases. The aim of this investigation was to study the effect of a number of newly synthesized coordination compounds of Ge(IV) and Ba(II), (Co(II), Ni(II), Cu(II), Zn(II) with gluconic acid on the activity of Penicillium tardum and Eupenicillium erubescens α-L-rhamnosidases. Methods. The objects of the study were Penicillium tardum and Eupenicillium erubescens α-L-rhamnosidases. α-L-Rhamnosidase activity was determined by the Davis method using naringin as a substrate. Coordination compounds Ge(IV) and Ba(II), Co(II), Ni(II), Cu(II),and Zn(II) with gluconic acid were used as enzyme activity modifiers. The synthesized complexes correspond to the formulas [M(H2O)6][Ge2(OH)2(C6H8O7)2]⋅nH2O (M = Ba(1), n = 2; Co 2, n = 4; Ni(3), n = 4; Cu(4), n = 4; Zn(5), n = 3). Results. The effect of coordination compounds 1-(5) on the activity of α-L-rhamnosidase in two strains of Penicillium tardum and Eupenicillium erubescens was studied depending on the exposure time and concentration of the effector. It was shown that compound (3) at a concentration of 0.01% (1 h incubation) led to a slight (by 5%) increase in the activity of P. tardum α-L-rhamnosidase. Compound 1 at a concentration of 0.1% led to a decrease in the activity of P. tardum α-L-rhamnosidase by 29% during the first hour, and after 24 h of incubation, a decrease in the inhibitory effect to 15% was noted. Compounds 2 and (4) activated the enzyme by 9-39% at 1h exposure. At a concentration of 0.1% and exposure time of 1 h, compound 1 increased the activity of E. erubescens α-L-rhamnosidase by 80%, while at a decrease in concentration to 0.01%, the activity increased only by 29%. In general, it should be noted that in most cases, an increase in the duration of incubation up to 24 h led to a decrease in the level of activation (or inhibition) and a return to the control values of enzyme activity. Conclusions. The variety of effects of metal coordination compounds on the activity of enzymes, depending on the nature of the cation and the origin of the enzyme, has been established. The involvement of Ba(II) had the greatest activating effect on the activity of E. erubescens α-L-rhamnosidase compared to other metals. [ABSTRACT FROM AUTHOR]

The intensive development of biotechnology in the last decade is largely determined by the growing requirement needs of both medicine and various industries for products of microbial synthesis, including glycosidases, in particular a-L- rhamnosidases. lheir wide use to solve current biological-medical and chemical-technological problems stimulates re- searchers to search for compounds capable of influencing their catalytic activity. Therefore, the purpose of this work was to isolate and purify a-L-rhamnosidase from a new producer of Penicillium restrictum and to investigate multi-ligand germanium-3d-metal complexes with citric acid, phenanthroline, and bipyridine as effectors of its activity. Methods. Ihe object of the study was a-L-rhamnosidase of restrictum. Its purification was carried out by gel filtration and ion exchange chromatography on TSK-gels and Sepharose 6B. The activity of a-L-rhamnosidase was determined us- ing the Davis method with naringin as a substrate. As modifiers of enzyme activity, purposefully synthesized multi- ligand germanium-3d-metal complexes with citric acid, phenanthroline, and bipyridine (INi(bipy)3[Ge(HCit)2] •3H 2 O (1); [Ni(phen)3 ] [Ge(HCit)2]•2H2O (2); [{Cu(bipy)2}2Ge(μ-Cit)2] •12H20 (3); [{Cu(phen)2]2Ge-(Cit)2]•13H2O (4); [Zn(bipy)3][Ge(HCit)2].•12H20 (5); [Zn(phen)3][Ge(HCit)2]•3H2O (6)), were used. Results. From the supernatant of culture fluid of P. restrictum, a-L-rhamnosidase was isolated and purified 23.1 times with a yield of 0.09%. the specific activity of the enzyme was 27.8 units/mL. The enzyme was homogeneous according to gel filtration on Sepharose 6B and had a molecular mass of 50 kDa. It was established that the considered coordination compounds are able to regulate the catalytic activity of a-L-rhamnosidase of P. restrictum. All of them manifest themselves either as a tivators or as inert substances, no inhibition was observed. In addition, the dependence of the degree of enzyme activation by the compounds on their concentration is traced and corresponds to the following series: at a concentration of 0.01% —1 > 6 =-- 5 > 3 >2 =.-- 4 and at a concentration of 0.1 % —1 > 4 > 2 > 5 = 6.3. The catalytic activity is also significantly affect-ed by the time of exposure to the compounds: at a concentration of 0.01% for lh, the activity of the enzyme at the control level was observed for all compounds, whereas at a concentration of 0.1% for 24 h,the activity increased sharply in the presence of compounds 1(300%), 6 (153%), and 2 (134%). The action of the others was at the control level. Conclusions. The obtained data on new complex metal compounds with an activating effect on microbial a-L-rhamnosidases. It has been established that compounds whose structural organization ensures the synergism of the action of all components are the most promising enzyme effectors in a series of coordination compounds of biologically active metals and ligands. [ABSTRACT FROM AUTHOR]

In recent years, the particular interest of researchers is focused on such enzymes as α-L-rhamnosidase and α-galactosidase. These enzymes are considered useful for various applications. α-L Rhamnosidases may be applied for debittering of citrus fruit juices, due to the less bitter taste of the derhamnosylated flavonones, for rhamnose production, and for the determination of the anomeric configuration in polysaccharides, glycosides and glycolipids. These enzymes may enhance wine aroma and flavonoid bioavailability, or assist in the synthesis of pharmaceuticals. α-Galactosidase finds application in many areas. It is widely used in the food industry to improve the quality of soy products by hydrolyzing indigestible galactosides such as raffinose and stachyose, in the processing of raw materials in order to increase the yield of sugar from molasses, and for the biotransformation of human blood erythrocytes of group B (III) in universal donor erythrocytes, as well as in enzyme therapy of some congenital disorders of sphingolipid metabolism. Earlier, as a result of screening microorganisms of different taxonomic groups, we has selected active α-L-rhamnosidase and α-galactosidase producers. One way to increase their activity is using various effector compounds capable of modifying the enzyme activity. The study of the influence of various effectors is one of the priority areas of modern research in biochemistry, biocoordination chemistry, and biotechnology. Recent advantages in the area of biocoordination chemistry revealed high activating properties of double heterometallic mixed-ligand coordination compounds with germanium(IV)/tin(IV) tartaric complex anions and 1,10-phenanthroline/2,2'-bipyridine d-metallic cations. The aim is to estimate the enzyme-effector activity of five similar tartratostannates for the α-L-rhamnosidases of Cryptococcus albidus, Eupenicillium erubescens, and α-galactosidase of Penicillium restrictum. Methods. The activity of α-Galactosidase was determined using p-nitrophenyl-α-D-galactopyranoside («Sigma», USA) as a substrate. The activity of α-L-rhamnosidase was determined using the Davis method. As modifiers of enzyme activity, [Fe(phen)3]2[Sn2(μ-Tart)2(Н2Tart)2]·2H2O (1), [Co(phen)3]2[Sn2(μ-Tart)2(Н2Tart)2]·8H2O (2), [Ni(phen)3]2[Sn2(μ-Tart)2(Н2Tart)2]·2H2O (3), [Cu(phen)3]2[Sn2(μ-Tart)2(Н2Tart)2]·2H2O (4), and [Zn(phen)3]2[Sn2(μ-Tart)2(Н2Tart)2]·6H2O (5) were used. Results. The study of the effect of complexes 1--5, which are supramolecular salts consisting of the same tartrate stannate anion (electrophilic agent) and a 1,10-phenanthroline d-metal cation (nucleophilic agent), on the Cryptococcus albidus, Eupenicillium erubescens α-L-rhamnosidases, and Penicillium restrictum α-galactosidase showed that the compounds tested had a different influence on the enzymes' activity. The catalytic activity of α-L-rhamnosidase is significantly influenced by all complexes. The effectiveness of compounds 1--5 for P. restrictum α-galactosidase was less pronounced in comparison with C. albidus and E. erubescens α-L-rhamnosidases. It was mostly at the control level. There was observed a certain pattern in the influence of complexes on α-L-rhamnosidases of Cryptococcus albidus and Eupenicillium erubescens. Compounds 2 and 5 turned out to be the most effective and activated enzymes by 500-900%. Conclusions. Compound 2 [Co(phen)3]2[Sn2(μ-Tart)2(Н2Tart)2]·8H2O is promising for further use as an effector of the α-L-rhamnosidase activity. [ABSTRACT FROM AUTHOR]

In recent years, cold-adapted enzymes are increasingly used in industrial processes such as the food, textile and beverage industries. Moreover, cold-active enzymes are usually thermolabile and can be inactivated with little heat. This is especially important in reactions where it is necessary to inactivate an enzyme after it has completed its function, while maintaining conditions that allow other enzymes involved in the reaction to function. Among these enzymes, glycosidases play an important role, which are used in medical technological processes, the food industry, biotechnology for the purification and processing of raw materials, as well as in many other areas of human activity. Therefore, the aim of this work was to study the ability of the psychrotolerant bacterium Pseudomonas mandelii U1 to produce glycosidases, in particular a-L-rhamnosidases, and also to investigate their physicochemical properties and substrate specificity. Methods. Glycosidase activities were determined by Romero and Davis methods, protein - by Lowry method. Results. The study of enzymatic activities in the dynamics of growth indicates that already on the third day of cultivation in the supernatant of the culture liquid of P. mandelii U1 α-L-rhamnosidase activity (0.09 U/mg protein) was noted. On the fifth day of cultivation, in addition to α-L-rhamnosidase (0.09 U/mg protein), β-D-glucosidase (0.09 U/mg protein) and α-D-glucosidase (0.09 U/mg protein) activities were identified. On the seventh and ninth days of cultivation, the spectrum of glycosidase activities was wider, except for α-L-rhamnosidase (0.2 and 0.16 U/mg protein, respectively), β-D-glucosidase (0.02 and 0.05 U/mg protein, respectively) and α-D-glucosidase (0.04 and 0.08 U/mg of protein, respectively), α-D-mannosidase (0.025 and 0.025 U/mg protein, respectively), α-D-fucosidase (0.025 and 0.05 U/mg protein, respectively), N-acetyl-β-D-glucosaminidase (0.025 and 0.025 U/mg protein, respectively) and N-acetyl-β-D-galactosaminidase (0.025 and 0.025 U/mg protein, respectively). Since among the studied glycosidase activities, α-L-rhamnosidase was the highest, subsequent studies were aimed at investigating its properties. It was shown that P. mandelii U1 α-L-rhamnosidase has the pH optimum of action at 5.0, and the temperature optimum - at 4 °C. Conclusions. The temperature optimum of P. mandelii U1 α-L-rhamnosidase preparation isolated from moss in Antarctica, Galindez Island, is 4 °C, the optimum pH is 5.0, the enzyme is able to hydrolyze as synthetic substrates p-nitrophenyl-α-L-rhamnopyranoside, p-nitrophenyl-β-D-glucopyranoside, p-nitrophenyl-α-D-glucopyranoside, p-nitrophenyl-α-D-mannopyranoside, and natural substrates - naringin, neohesperidin and rutin, which suggests the possibility of its use in the future in food technologies, in particular in food processing and waste degradation at low temperatures. [ABSTRACT FROM AUTHOR]

α-L-Rhamnosidase [EC 3.2.1.40], enzyme of the hydrolase family has a wide range of applications: in the food industry, for example, in winemaking to improve the quality and aroma of wines, in the production of citrus juices and drinks to remove bitter components (naringin) that improves the quality and nutritional value of these products; in research as an analytical tool for studying the structure of complex carbohydrate-substituted biopolymers. For the successful use of α-L-rhamnosidases in various biotechnological processes, an important aspect is the development of ways to increase their activity. The main factors affecting the growth and metabolism of microorganisms, including the synthesis of enzymes, are the physicochemical conditions of cultivation, the composition of the nutrient medium, the introduction of substances that raise the yield of the enzyme, which is manifested in an increase in its activity. At present, one of the priority directions of modern research is the study of the effect of various effector compounds that are capable to modify the studied enzymatic activity. In this work, which is a continuation of previous studies, a number of mixed-ligand and mixed-ligand-different-metal coordination germanium compounds of with xylaric acid (H5Xylar), 1,10-phenanthroline (Phen), 2,2-bipyridine (bipy) and ions of 3d-metals (Fe2+, Ni2+, Cu2+, Zn2+) were selected as effectors. Study of the effect of these complexes on the activity of Eupenicillium erubescens, Cryptococcus аlbidus and Penicillium tardum α-L-rhamnosidases were the aim of this work. Materials and methods. The objects of research were α-Lrhamnosidases from Eupenicillium erubescens 248, Cryptococcus albidus 1001, and Penicillium tardum IMV F-100074. The α-L-rhamnosidase activity was determined by the Davis method using naringin as a substrate. We used 12 coordination compounds of germanium as modifiers of enzyme activity, the composition and structure of which were established using a combination of physical and chemical research methods: elemental analysis, thermogravimetry, IR spectroscopy and X-ray structural analysis. Structures of seven compounds are deposited in the Cambridge Crystallographic Database. When studying the effect of various compounds on the activity of enzymes, concentrations of 0.1 and 0.01 % were used, exposure times were 0.5 and 24 hours. The test compounds were dissolved in 0.1 % dimethyl sulfoxide. UV-spectra of absorption of native and chemical modified preparations of the enzymes were studied by spectrophotometer-fluorimeter DeNovix DS-11 in the range of 220-340 nm, concentration of the enzyme preparation 1.0 mg of protein/mL. Results. Analysis of the totality of the obtained data (exposure time 24 h, concentration 0.1 %) regarding the effect of the studied compounds on the activity of E. erubescens, C. albidus and P. tardum α-L-rhamnosidases showed that the influence of the studied modifiers for the activity of α-L-rhamnosidases varies depending on the producer strain. Our data allow us to present the following series of modifiers in accordance with an increase in their effect on the activity of enzymes of different producers: E. еrubescens: 12 < 11 < 5 < 3 < 4=10 < 1 < 3 < 8 < 2 < 6 < 7; C. albidus: 10 < 11 < 12 < 9 < 3 < 1=5 < 8=4 < 2 < 6 < 7; P. tardum: 12=2 < 3 < 4 < 11 < 5 < 8 < 1 < 9 < 6 < 10 < 7. Conclusions. The results obtained allow us to conclude that compound (7)(-tris(bipyridine) nickel(II) μ-dihydroxyxylaratogermanate(IV)) is the most effective activator of α-L-rhamnosidases of all three micromycete strains, compound (6)(tris(phenanthroline)nickel(II) μ-dihydroxyxylaratogerman ate(IV)) - on α-L-rhamnosidase from E. erubescens and C. albidus, while compound (10)-(copper(II) μ-dihydroxyxylaratogermanate(IV)-cuprate(II)) - only of P. tardum α-L-rhamnosidase. [ABSTRACT FROM AUTHOR]

The phylogenetic structure of bacteria is not always consistent with the traditional classification scheme based on the phenotypic properties of bacteria. That is one of the problems of modern bacterial taxonomy. In addition, traditional methods to identify bacteria using phenotypic properties have a number of disadvantages. In recent decades, significant progress was achieved in the investigation of microbial world using molecular methods for fast identification. The aim of the study was to clarify the species status of four strains of bacteria isolated from black lichens of the cliffs of Galindez Island in the Antarctic on the basis of phenotypic and genetic analysis. Methods. Morphological and cultural properties of bacteria were studied according to generally accepted microbiological methods. Physiological and biochemical ones were investigated using test systems API Coryne and API 20E (bioMerieux SA, France), according to the manufacturer's instructions. Phylogenetic analysis was performed based on the nucleotide sequences of the 16S rRNA gene. To identify closely related species, a comparative analysis of the nucleotide sequences of 16S rRNA genes was performed using the BLAST software package. The phylogenetic position was determined by constructing trees (dendrograms) to show the position of the studied strains among closely related and typical species (programs ClustalX 2.1, Mega 6.06). The tree was constructed applying ClustalX 2.1 by comparing the nearest neighbors with bootstrap analysis (bootstrap NJ tree) using 1000 bootstrap tests (1000 alternative trees). Then the phylogenetic tree was edited by the program Mega v. 6.00. Results. Based on the results of comparative, phylogenetic and phenotypic analysis, the studied Antarctic strains 180n1, 181n2, 188n2, 190n2 were identified as Pseudomonas fluorescens, Microbacterium foliorum, Sporosarcina aquimarina and Rothia sp., respectively. The coefficient of similarity of 16S rRNA genes of strain 180n1 with such a closely related species from the database P. fluorescens NBRC 14160 was 99.5%; 181n2 with M. foliorum P 333/02 - 99.4%; 188n2 with S. aquimarina SW28 - 99.7%. These strains form common clusters with closely related species on phylogenetic dendrograms. The strain 190n2 can be considered as Rothia sp., since has the remote position from closely related strains in the cluster Rothia and a low percentage of similarity (97.3%) with the species Rothia endophytica YIM 67072. These strains belong to the phyla: Firmicutes, Actinobacteria, Proteobacteria. Conclusions. Phylogenetic and phenotypic analyzes allowed determining the taxonomic position of isolated aerobic chemoorganotrophic microbial strains of the Antarctic. Nucleotide sequences of 16S rRNA genes are deposited in the International GenBank database under numbers HG518622, HG518623, HG518625, HG518626. [ABSTRACT FROM AUTHOR]

The directed search for extremophilic producers in order to obtain hydrolytic enzymes with increased thermal stability has an unconditional practical potential for use in the food and feed industry to improve the quality of the final product. The aim of the work was to study the ability of collection strains of thermophilic fungi to show α-L-rhamnosidase, α-galactosidase, cellulase, β-mannanase, keratinase and caseinolytic activity. Methods. Micromycetes were grown under submerged conditions in test tubes at 42 °C for 8-14 days. Enzymatic activities were studied in the culture liquid supernatant. p-Nitrophenyl-α-D-galactopyranoside, naringin, guar gum galactomannan and Na-carboxymethylcellulose were used as substrates to determine α-galactosidase, α-L-rhamnosidase, β-mannanase and cellulase activities, respectively. Casein and crushed defatted feathers were served as substrates for the determination of proteolytic activity. Results. The enzymatic activity of 50 strains of micromycetes belonging to 17 species was investigated. The studied group showed high activity: 94 % of the strains had at least one, 34 % - two, 26 % - from three to five enzyme activities. The most active keratinase producers were Thielavia terrestris 1920 and 62, Rhizomucor tauricus 1909, Chrysosporium thermophilum 2050, Thermoascus thermophilus 92 and Thermoascus aurantiаcus 2052 (10-26 U/mL). The highest α-L-rhamnosidase activity was observed in T. terrestris 62 (0.35 U/mL), and carboxymethylcellulase activity -in Thermomyces lanuginosus 2046. Six strains showed α-galactosidase (0.05-0.2 U/mL) and four strains - β-mannanase (5-130 U/mL) activity. Conclusions. As a result new strains producing proteolytic and glycolytic enzymes were isolated among thermophilic micromycetes. Soil thermophilic micromycetes can be used as producers of proteolytic and glycolytic enzymes. Of particular interest are the cultures of Acremonium thermophilum 1963, Corynascus thermophilum 2050, C. sepedonium 1899 and 65068, T. thermophilus 1946, which are capable of producing complexes of proteases and glycosidases in the culture liquid. This indicates that these strains are promising for use as destructors in various technologies processing of complex raw materials. [ABSTRACT FROM AUTHOR]

The Antarctic region has significant potential to study the biodiversity of microorganisms and to search for bacterial producers of glycolytic and proteolytic enzymes with new properties. The aim was to study the extracellular glycosidase and proteolytic activity of four bacteria strains isolated from black lichens of the cliffs of Galindez Island in the Antarctic. Methods. Cultures of bacteria were grown in submerged conditions at a temperature of 15 and 26 oC for 48 h. The synthetic and natural substrates such as p-nitrophenyl-glycosides, soluble starch, gelatin, casein and Elastin-Congo red were used to study the enzymatic activity of bacteria. Results. All studied strains showed α-fucosidase activity. Microbacterium foliorum, Sporosarcina aquimarina and Rothia sp. exhibited α-, β-xylosidase, β-glucosidase or β-N-acetylglucosaminidase activity in different ratios. That may indicate the presence of the enzymatic complex of hydrolysis of lichenan and xylan, which are part of polysaccharides of plants and lichens. P. fluorescens and M. foliorum also showed gelatinase activity. The enzymatic activity of bacteria was noted to be higher in the case of cultivating at 15 oC compared to 26 oC. The α-xylosidase of M. foliorum was optimally active at pH 6.0 and 35 oC, the α-xylosidase of Rothia sp. - at pH 6.5 and 35 oC. High level of stability was shown for these enzymes in the pH range from 4.0 to 7.0 and temperature from 15 to 35 оС during 24 h. Conclusions. Antarctic lichens can be a source of bacterial producers of polysaccharide degrading enzymes with new properties and low temperature optimum. The Antarctic cold environment provides the great opportunities to study the adaptive mechanisms of microorganisms and their enzymatic systems in order to develop new biotechnologies. [ABSTRACT FROM AUTHOR]

The aim of the work was to investigate the practical using of α-L-rhamnosidase substrate specificity Cryptococcus albidus. p-Nitrophenyl derivatives of monosaccharides were used to determine the activity and the enzyme specificity. The ability to hydrolyze of natural substrates was evaluated by Davis and high-performance liquid chromatography methods. It was shown that the enzyme exhibited narrow specificity towards the glycon of synthetic substrates and hydrolyzes only p-nitrophenyl-α-Lrhamnopyranoside (Km 4.5 mM) and p-nitrophenyl-α-D-glucopyranoside (Km 10.0 mM). C. albidus α-Lrhamnosidase the most active degrades naringin (Km 0.77 mM), releasing prunin and naringenin. Km for neohesperidin was 3.3 mM. The efficacy of the naringin hydrolysis in grapefruit and pomelo juice was 98 and 94% in 60 min (40 °C, 2 U/ml). As the result of of green tea and orange juice treatment by α-Lrhamnosidase, there was a decrease in the content of rutin, narirutin and hesperidin, indicating that α-1,2-and α-1,6-linked rhamnose could be cleaved from natural flavonoids. The study shows the possibility of citrus juices and green tea treatment by C. albidus α-L-rhamnosidase for the purpose of their taste qualities improvment and obtaining bioavailable flavonoids glucosides. [ABSTRACT FROM AUTHOR]

The aim of the research was a comparative study of the thermal stability of native and modified by various methods α-L rhamnosidases of Eupenicillium erubescens and Cryptococcus albidus for improving the stability of enzymes. Denaturation of native and modified enzymes were performed at 65 0C, pH 5.2. Enzyme activity was determined using p-nitrophenyl-α-L-rhamnopyranoside and naringin. It is found that in the treatment by polyethylenglycol PEG 1500, dextrans 500 and 70 T thermostability of α-L-rhamnosidases tested decreases, while modification with polyethylenglycol 20000 leads to increase thermal stability of the E. erubescens enzyme to 280% and C. albidus to 150%. Comparative study of the thermal stability of the native and modified by cellulose and its derivatives α-L-rhamnosidases of C. albidus and E. erubescens showed that at concentrations cellulose of 5-15 μg/10 μg protein protective effect of polymers on enzymes was investigated was observed. Hydrophobic modifications using succinic anhydride also can slow down the denaturation of α-L-rhamnosidases tested under experimental conditions. These stabilized C. albidus and E. erubescens α-L-rhamnosidases can be used in biotechnological processes. [ABSTRACT FROM AUTHOR]

The study of the features of thermal inactivation of α-L-rhamnosidase from Eupenicillium erubescens aiming to develop further strategies for improving the stability of the enzyme was the purpose of the research. Two forms of α-L-rhamnosidase were studied. They were obtained by producer cultivation on two different carbon sources (rhamnose and naringin). It was shown that at temperatures above 60°C heat inactivation process is described by the first order reaction kinetics. It was established that stability of α-Lrhamnosidase from E. erubescen, grown on naringin depends on hydrophobic interactions rather than electrostatic ones. It was shown that aggregation of enzyme molecules could lead to their thermal stabilization. The important role of cysteine residues and possible involvement of the metal cation in maintaining of active form of α-L-rhamnosidases from E. erubescens was highlighted. [ABSTRACT FROM AUTHOR]

The aim of research was to investigate the prevalence of complex mannan-degrading enzymes among museum and freshly isolated cultures of micromycetes, actinobacteria and bacteria. It has been shown that the producers of β-mannanases mostly extracted from sources that are rich in plant residues. We detected strains of Penicillium aculeatum, P. tardum and P. rugulosum which produce a complex of β-mannanases and α-galactosidase activity. Also, strains of thermophilic micromycetes species Corynascus sepedonium, Scytalidium thermophilum and Rhizomucor tauricus with high mannanase activity in culture liquid were detected (10 – 130 U/ml). Two strains of P. aculeatum and P. tardum showed β-mannanase, β-mannosidase and α-galactosidase activity. Actinobacteria was shown high potential, 70 % of all tested strains showed mannanase activity; their activity ranged from 2 to 55 U/ml. Among the most active bacterial cultures were representatives of soil microflora: Bacillus circulans, B. subtilis, B. mesentericus, where as among the strains isolated from sea water the active mannanases producers were not found.

The effect of cations, anions and specific chemical reagents: 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide methiodide, EDTA, o-phenanthroline, dithiotreitol, L-cysteine, beta-mercaptoethanol, p-chlormercurybenzoate (p-ChMB), N-ethylmaleimide on the activity of alpha-L-rhamnosidase of Eupenicillium erubescens has been investigated. The essential role of Ag+ and Hg2+ which inhibit the alpha-L-rhamnosidase activity by 47-73% has been shown. Whereas L-cysteine exhibits the protective effect, rhamnose in concentration of 1-5 mM does not protect the enzyme from negative effect of Ag+ and Hg2+. Basing on the inhibitory and kinetic analysis it was supposed that the carboxyl group of C-terminal aminoacid and imidazole group of histidine take part in the catalytic action of alpha-L-rhamnosidase. It was assumed that sulphydryl groups took part in catalysis, carried out by alpha-L-rhamnosidase of E. erubescens, since the activity of alpha-L-rhamnosidase inhibited by p-ChMB and thiol reagents such as dithiothreitol, L-cysteine, beta-mercaptoethanol did not remove its inhibitory action.

Investigation of 15 different glycoside activities of 64 strains isolated from water and invertebra of the Black Sea has shown that 64% of the studied strains displayed the capacity to synthesize enzymes with alpha-L-ramnosidase activity which varied from 0.01 to 0.20 un/ml depending on the strain. The greatest number of the enzyme producers was found in representatives of Alteromonas macleodii. Other investigated glycosidase activities: alpha-amylase, beta-N-acetyl-D-glucosaminidase, beta-D-glucuronide, alpha-N-acetyl-D-galactosaminidase, beta-N-acetyl-D-galactosaminidase, beta-D-galactosidase, alpha-D-galactosidase, beta-D-glucosidase, KM-cellulase activities though have been found, but mainly with inconsiderable indices, alpha-D-glucosidase, alpha-D-mannosidase, alpha-L-fucosidase, beta-D-xylosidase and alpha-D-xylosidase activities were found in neither of the studied strains. Strains with rather high proteolytic activity were found among marine species of bacteria. It has been established that 18 strains (28%) of 64 marine isolates were characterized by rather high level of total proteolytic activity (from 0.1 to 05 un/ml), 43.75% of them displayed inconsiderable (up to 0.1 un/ml) or only trace (up to 0.01 un/ml), 18.75% did not display any hydrolytic activity in respect of casein. Investigation of substrate specificity to a number of fibrillar and globular proteins of 9 studied strains, which displayed considerable general (caseinolytic) activity has shown that 8 of them displayed fibrinolytic activity from 0.15 to 2.175 un/ml. All 9 strains were characterized by gelatin activity. Collagenase and keratinase activity was also revealed. Neither of 9 studied strains displayed elastase activity.

Influence ofsome technological parameters of cultivation ofproducers Cryptococcus albidus, Eupenicillinum erubescens, Bacillus sp. on the process of synthesis of extracellular enzyme alpha-L-rhamnosidase has been studied. The authors have determined optimal sources of carbon (0.2-0.3% rhamnose) and nitrogen (0.2% sodium nitrate for C. albidus and E. erubescens and ammonium sulphate for Bacillus sp.) (the ratio 1:2), cultivation temperature (28 degrees C, 25 degrees C, 42 degrees C, respectively) for maximum synthesis of alpha-L-rhamnosidase. Use of the medium with initial pH value from 4 to 8 was most efficient for all the studied strains. The maximum level of alpha-L-rhamnosidase activity of E. erubescens and Bacillus sp. was established at the value of sulphite number of 0.44, while for C. albidus--it was 0.56. Maximum alpha-L-rhamnosidase activity of C. albidus, E. erubescens, Bacillus sp. is achieved at 4, 8 days and 27 hours of cultivation, respectively. The cultures being grown in selected conditions, the alpha-L-rhamnosidase synthesis has increased by 30, 50 and 20%, respectively.

The influence of a number of coordinative compounds of zinc with N-substituted thiocarbamoil-N'-pentamethylensulfenamides on activity of elastase, alpha-L-rhamnosidase and alpha-galactosidases evidence for a possibility of their usage as stimulators or inhibitors of enzymes tested have been studied. It was shown that all the compounds in concentration of 0.1 and 0.01% inhibited by 90-100% Bacillus thuringiensis 27-88Els+ elastase activity. [Zn(L2)Br2], [Zn(L1)(NCS)2] and [Zn(L3)(NCS)2] at 20 h exposition activated Cryptococcus albidus 1001 alpha-L-rhamnosidase activity. The rest of compounds influenced it on the control level or inhibited it by 7-23%. The obtained results testify that essential role is not played by separate fragments (L-ligand and anions), but by molecules of zinc complexes as a whole. All the studied complexes, exept for [Zn(L3)(NCS)2], induced alpha-L-rhamnosidase activity of Eupenicillium erubescens 248 (7 to 60%). All zinc compounds (concentration 0.01%, exposition time - 60 min) influenced at the control level Aspergillus niger and Cladosporium cladosporioides alpha-galactosidases activity, however inhibited (up to 20%) activity of Penicillium canescens alpha-galactosidase. The increasing of exposition time of the compounds tested with enzymes up to 20 h testify to selective action of separate compounds on enzymes tested. The data obtained prove, that the character of interaction of zinc complexes is changed depending on the enzyme tested and its strain-producer.

The kinetics and mechanism of thermal inactivation of Penicillium canescens alpha-galactosidase in the temperature range of 55-65 degrees C have been studied. The kinetic scheme of alpha-galactosidase thermal inactivation was proposed which included the reversible dissociation of active hexamers into associating monomers and irreversible denaturation of monomers. The kinetic constants of thermal inactivation have been determined. The effect of enzyme concentration and purification efficiency has been investigated. A possibility of defence of protein molecule from thermal inactivation in the presence of BSA, glycerol, melibiose, raffinose and stachyose is shown.

The influence of different factors on biosynthesis of extracellular alpha-amylase of Aspergillus sp. 55 grown at submerged cultivation has been studied. The optimal composition of nutrient medium (1 g of starch and 0.5 g of NaNO3 per 1 l) was chosen. The enzyme preparation has a wide pH optimum of activity (4.5-9.0), thermooptimum at pH 6.5 was 60 degrees C, at pH 4.5 and 9.0--50 degrees C. The inhibitory effect of coordinative germanium compounds on amylolytic activity of the preparation was shown. The paper is presented in Ukrainian.

The current data on microbial mannanases are summarised in the work. The questions concerning the spreading of mannanases substrates--mannans in nature, their structural variations are regarded. The production of mannanases by micro-organisms of different taxonomic groups, their physico-chemical properties and substrate specificity are described. The ways of mannans hydrolysis by mannanase are estimated. The questions of cloning and expression of mannanase genes have been discussed. These results are very important for producing highly effective microbial biosynthetics.

On the basis of the data of metal ions influence it was established that Penicillium commune 266 alpha-L-rhamnosidases are dependent of metals. The cations of Cu2+, Mn2+, Ag+, Hg2+ essentially influenced the activity decreasing its up to 45-55%. Ba2+ and Mn2+ ions completely inhibited alpha-L-rhamnosidase 2 activity in concentration of 10(-3) M. The role of carboxylic group and histidine imidazole group in hydrolysis of rhamnoglycosides have been estimated by the Dixon, Lineweaver-Burke and photooxidation methods. It was also shown that sulfhydrilic groups which do not take part in catalysis, but are necessary for displaying the enzymatic activity are located at a certain distance from P. commune 266 alpha-L-rhamnosidase active centre.

Antihypertensive therapy with sequential addition of drugs (noliprel, noliprel-forte, metoprolol, amlodipine) for achievement of target blood pressure (BP) below 140/90 mm Hg was used in the treatment of 99 patients with arterial hypertension (AH) with (n=51) or without left ventricular (LV) hypertrophy (LVH). At initial Doppler study of transmitral blood flow all patient with LVH had type 1 (n=48) or type 2 (n=3) diastolic LV dysfunction. Among patients without LVH 13 had minor type 1 diastolic LV dysfunction. After 12 - 14 months of antihypertensive therapy in all 44 patients with moderate LVH (myocardial mass index below 140 g/m2) BP corresponded to target level. This was associated with 6% decrease of myocardial mass index (MMI) and its normalization in 2/3 of patients, restoration of diastolic function in 3/4 of patients and its improvement in other patients, decrease of functional class, in rease of 6 min walking distance, and improvement of quality of life according to questionnaire for patients with CHF. In 7 patients with pronounced LVH (MMI 140 g/m2) target BP was not achieved, LVMMI, diastolic function, and functional class did not change, however tolerance to physical effort and quality of life improved. Thus in all patients with AH without LVH target BP level was achieved. In minor initial diastolic dysfunction diastolic function restored to normality, functional class, tolerance to physical work and quality of life improved.

alpha-N-acetylgalactosaminidase has been isolated from liquid culture of micromycete Aspergillus niger and purified 600 times by ammonium sulphate precipitation followed by ion exchange and gel-filtration chromatography on TSK-gels with specific activity 10.5 U/mg of protein. The preparation was homogenic: its molecular mass by the data of gel-filtration on Sepharose 6B was 430 kDa, on PAAGE in the system of DDSNa--70 kDa. That gives every reason to suppose oligomeric structure of the enzyme molecule. The carbohydrate component, including mannose, galactose, glucosamine and two nonidentified hexosamines was observed in alpha-N-acetylgalactosaminidase. Thermo- and pH- optima were 60 degrees C and pH 3.5, respectively. The enzyme was thermo- and pH-stable, resistant in storage. The enzyme was found to exhibit strict specificity in respect ofglycon. It was shown that enzyme was competitively inhibited by substrate and reaction product. Km and Vmax with respect to nitrophenyl substrate were 1.25 mM and 10.5 mkmole/min/mg of protein. The activity of glycosidase tested was independent of the presence of metal ions. The presence of carboxylic group of C-terminal aminoacid and imidazol group of hystidine in active centre of molecule was established. A number of natural and synthetic substrates were able to activate (50-200%) production of A. niger alpha-N-acetylgalactosaminidase.

Screening of producers of alpha-L-rhamnosidase among 692 strains of microorganisms of different taxonomic groups has been performed. A capacity to synthesize the enzyme was revealed in 35.7% of the studied cultures. Representatives of genera Aspergillus, Myrothecium, Penicillium, Eupenicillium (activity 0.1 - 0.335 un./mg of protein) proved to be the most active producers. Complex preparations of alpha-L-rhamnosidase were obtained from cultural liquid of 10 producers by fractionation with ammonium sulfate (30 and 90% saturation); pH- and thermal optimum, as well as pH- and thermostability were analyzed in 7 of them. The Aspergillus, Penicillium, Eupenicillium strains which display high stability and activity under technological values of temperature 20-37 degrees C and medium pH 4.0-6.0 have been chosen for further investigations.

It was established, as a result of investigation of kinetic properties of two fungal glycosidases (alpha-N-acetylgalactosaminidase and alpha- galactosidase), that Km and Vmax for the corresponding synthetic substrates were 1.19 and 1.25 mM, 25.0 and 10.5 micromol/min/mg of protein, respectively. a-Galactosidase and alpha-N-acetylgalactosaminidase were also competitively inhibited by the reaction products - D-galactose and N-acetylgalactosamine, the inhibition constants were 8.5 x 10(-3) and 6.2 x 10(-2) M, respectively. One could also observe the inhibition of alpha-galactosidase reaction in the presence of 10(-3) M of fucose, lactose and arabinose. It was shown that the rate of enzymatic hydrolysis of nitrophenyl substrates was directly proportional to the concentration of enzymes, and the increase of the substrate concentration leads to the increase of hydrolysis rate. The substrate concentration being increased above the optimal one (3.6 mg/ml), the decrease of the reaction rate owing to the formation of inactive enzyme-substrate complex FS2 may be observed.

Result of author's research and data from literature have been generalized with respect to hydrolase and transferase activity of glycosidases: alpha-galactosidase and alpha-N-acetylgalactosaminidase--the enzymes which catalyse hydrolysis of natural and synthetic glycosides. Broad variability of action specificity of glycosidases with respect to glycon, aglycon as well as the bond type depending on the enzyme isolation source have been shown. One can suppose that the enzyme action specificity is connected with different formation mechanisms of enzyme-substrate complexes. An idea is discussed concerning the identity of the mechanism of splitting of various glycosidic links by the studied enzymes.

Activity of alpha-N-acetylgalactosaminidase and alpha-galactosidase isolated from the culture medium of micromycete Aspergillus niger v. Tiegh F-16694 has been studied as affected by anions, cations and specific chemical reagents (n-chlormercurybenzoate, L-cysteine, dithiotreitol, beta-mercaptoethanol, EDTA, o-phenanthroline, sodium azide, hydrogen peroxide). It has been established that silver ions noncompetitively inhibit alpha-galactosidase at pH 5.2, the inhibition constant (Ki) being 2.5 x 10(-4) M. Galactose in concentration of 1-5 mM does not protect the enzyme from the negative action of silver ions, but this inhibitory effect is almost completely removed by the corresponding concentrations of L-cysteine. The same noncompetitive character was inherent in the inhibition of alpha-galactosidase reaction by mercury ions and n-chlormercurybenzoat (Ki is 4.5 x 10(-6) and 1.8 x 10(-4), respectively). The importance of sulphydryl groups for the support of active comformation of alpha-galactosidase molecule was established on the basis of inhibition and kinetic analysis. It has been shown that the enzyme molecule does not contain the groups which include metal atoms.

A scheme has been developed for isolation and purification of the enzyme with alpha-N-acetylgalactosaminidase and alpha-galactosidase activities which included fractionation by ammonium sulphate and chromatography on TSK-gels Toyopearl HW-60 and Fractogel DEAE-650-s and Sepharose 6B. The enzyme was purified 600 times with the yield of 28%. The enzyme preparation did not contain fucosidase, invertase and proteolytic activities. Molecular mass of the enzyme from the data of gel-filtration on Sepharose 6B was 430 kDa, according to the data of electrophoresis in DS-PAAG--70 kDa. It is shown that acidic and hydrophobic aminoacids prevail in the enzyme molecule, the carbohydrate component containing galactose, mannose, glucosamine and two nonidentified hexosamines is also present there. The enzyme preparation is stable during 48 hours at 20 degrees C; its pH-optimum is at pH 3.5-4.1. Michaelis constants concerning n-nitrophenyl-alpha-N-acetylgalactopyranoside and n-nitrophenyl-alpha-D-galactopyranoside were 1.18 and 1.25 mM, respectively.

Germanium complexes (IV) with succinic (H2Suc), oxyethyliminodiacetic (H2Oeida) and iminodisuccinic (H4Ids) acids as well as homo- and heteroligand germanium complexes (IV)--products of interaction of triammonium salt of oxyethylidendiphosphonic acid ((NH4)3HL) and oxyacids: tartaric (H4Tart), citric (H4Citr), trioxyglutaric (H4Toglut) acids have been synthesized. Composition of the obtained complexes: [Ge(OH)2(NaSuc)2].2H2O (I); [Ge(OH) (Oeida).H2O].H2O (II); [Ge(OH)2(NaHIds)2] (III); [Ge(OH)2(NH4)3HL) (H2Tart)] (IV); [Ge(OH)2(NH4)3HL) (H2Citr)] (V); [Ge(OH)2(NH4)3HL) (H2Toglut)] (VI); [Ge(OH)2((NH4)2HL)2] (VII); [Ge (OH)2((NH4)2HL)2] (VII); [Ge(OH)2 (H2O)2(NH4) HL] (VIII) has been determined. The capability of the synthesized compounds has been studied to affect synthesis and activity of the following enzymes: collagenase, alpha-N-acetylgalactosaminidase (alpha-GalNAc-ase) and alpha-galactosidase (alpha-Gal-ase). It has been established that the complexes II-VIII activate biosynthesis of alpha-Gal-ase and alpha-GalNAc-ase, while germanium dioxide (IX) and complex I possess considerable inhibiting effect on synthesis of the above enzymes. It has been also established that all the compounds except for IV increased the activity of both alpha-Gal-ase and alpha-GalNAc-ase. All the considered complexes demonstrated similar reaction with respect to collagenase: they inhibited both synthesis and activity.

Different factors have been investigated for their effect on the process of biosynthesis of alpha-N-acetylgalactosaminidase and alpha-galactosidase of Aspergillus niger under deep-water cultivation. Optimal sources and concentrations of carbon (soy flour--25 g/l) and nitrogen (pepton--7.5 g/l) were estimated. It has been established that temperature of 25 degrees C, pH 6.0, growing in 50 ml medium at the swing velocity 220 rev/min. for 6-8 days are optimal cultivation parameters. It has been shown that dry borine blood (in concentration of 1%) and a number of guanidine derivatives (guanidine carbonate--0.25%, nitroaminoguanison dimethylaminobenzaldehyde--0.05%, nitroaminoguanison of salicylic aldehyde--0.1%) can play the part of inducers of the mentioned glycosidase synthesis. When growing fungal culture in selected conditions synthesis of enzymes increased almost 3 times. Activity of alpha-N-acetylgalactosaminidase was 0.46 E/ml, and alpha-galactosidase--1.9 E/ml.

Screening of producers of alpha-N-acetylgalactosaminidase among 854 strains of micromycetes, 171 yeast and 357 bacterial cultures has been carried out. A capacity to synthesize the enzyme was revealed in 11% of cultures. Representatives of Aspergillus genus (activity--0.11-0.142 un./ml) were most active in producing the enzyme. It has been established that glucosidases spectrum in the culture liquid of 18 most active strains was characterized by complete homogeneity and by the presence of a rather high level (0.5-0.9 un./ml) of alpha-galactosidase activity. Complex preparations of alpha-N-acetylgalactosaminidase and alpha-galactosidase have been obtained from the culture liquid of producers by fractionation by ammonium sulphate (30 and 90% saturation); their pH- and thermo-optimum, pH- and thermal stability have been studied. It was shown possibility to induce alpha-N-acetylgalactosaminidase synthesis by a number of carbohydrates (galactose, glucose, galactosamine, and glucosamine), complex-forming substances (guanidine HCl), nitroaminoguanidin and guanidine carbonate and bovine blood. As a result the strain of Aspergillus niger 185 III was chosen which activity level could be increased more than 3 times (activity--0.6 un./ml as compared to initial one).