The influence of coordinaTive TarTraTe and malaTogermanaTe compounds on The acTiviTy of α-l-rhamnosidase preparaTions from Penicillium tardum, Eupenicillium erubescens and Cryptococcus albidus

recently enzyme preparations of microbial origin become increasingly important in different industries. Preparations of α-L-rhamnosidase are used in the pharmaceutical industry as well as in scientific work as a tool for analytical research. We have obtained purified α-L-rhamnosidase preparations from Penicillium tardum, eupenicillium erubescens and Cryptococcus albidus microorganism strains which are effective enzyme producers. the aim of the study was to estimate the ability of germanium coordination compounds to enhance enzyme catalytic activity. the effects of 11 heterometal mixed ligand tartrate (malate-)germanate compounds at 0.01 and 0.1% concentration on the activity of α-L-rhamnosidase preparations from Penicillium tardum IMV f-100074, eupenicillium erubescens and Cryptococcus albidus 1001 were studied at 0.5 and 24 h exposition. the inhibitory effect of [ni(bipy)3]4[{Ge2(oh)2(tart)2}3Cl2]·15h2 on P. tardum α-L-rhamnosidase was revealed. all studied compounds except [CuCl(phen)2][Ge(oh)(hMal)2] were shown to increase activity of P. tardum α-L-rhamnosidase at a longer term of exposition. activity of e. erubescens α-L-rhamnosidase was shown to be stimulated by d-metal cation-free compounds. C. albidus α-L-rhamnosidase occurred to be insensitive to all compounds studied.

I n recent years, enzymes of microbial origin are becoming increasingly important in food, pharmaceutical and chemical industries. Glycosidases, enzymes of the hydrolase family (O-glycoside hydrolases), which catalyze the hydrolysis of O-glycosidic bonds in glycosides, oligo-, polysaccharides, glycolipids and other glycoconjugates are of particular interest to researchers. Among them is α-Lrhamnosidase (α-L-rhamnoside-rhamnohydrolase, EC 3.2.1.40), which hydrolytically cleaves the terminal unreduced α-1,2, α-1,4 and α-1,6 bound residues of L-rhamnose in natural products such as naringin, rutin, quercetrin, hesperidin and other rhamnosecontaining glycosides [1][2][3]. α-L-Rhamnosidase 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. However, today, preparations of α-L-rhamnosidase are not available in Ukraine, and the high price of foreign commercial enzyme products (USA) significantly impedes their use in industrial technologies in Ukraine. With this in mind, we searched for effective producers of highly specific α-L-rhamnosidases among the strains of doi: https://doi.org/10.15407/ubj92.04.085 micro organisms of various taxonomic groups -bacteria, micromycetes, and yeast from the depositary of IMV, NAS of Ukraine. We selected the most active strains -Penicillium tardum [4], eupenicillium erubescens [5] and Cryptococcus albidus [6] and obtained α-L-rhamnosidase preparations from their culture fluids. Then we studied their physiochemical properties, substrate specificity, and functional groups involved in the catalytic process [7][8][9]. To obtain highly productive microbial enzymes, e.g. α-Lrhamnosidases, there are several approaches, one of which is the use of substances that enhance their catalytic effect. As α-L-rhamnosidase modifiers , coordination compounds of germanium, an essential ultratrace element, with various biologically active ligands have received increasing interest recently [10][11][12]. Due to the peculiarities of electronic configuration, germanium is able to promote tissue oxygenation, enhance blood circulation, strengthen the immune system, promote γ-interferon induction, normalize metabolism, and exhibit antitumor effect [13][14][15][16]. A lack of germanium can cause pathological conditions linked to reducing in immunity. Coordination compounds of germanium, which are able to increase the activity of α-L-rhamnosidases, may be useful in the development of therapeutic compositions based on rhamnose-containing compounds. Earlier [17], we obtained germanium complexes with citric acid and studied their effect on the α-Lrhamnosidase activity in enzyme preparations from fungi. All complexes were found to exert an activating effect, in contrast to the inorganic salts of Cu 2+ , Ni 2+ , Fe 2+ , Zn 2+ , Pb 2+ , Hg 2+ , which acted as inhibitors [18,19].
In this work, we continued our previous research and tested as effectors mix-liganded hetero-metal coordination compounds consisting of complex cations and anions [20][21][22]. Most of them contain central atoms-acceptors "metals of life": Cu, Ni, Zn, Fe, "essential" Ge and ligands of various types: nitrogen-containing heterocyclic bis-chelating antibacterial agents 1,10-phenanthroline, 2,2′-bipyridine and biologically active agents tartaric and malic acids with hydroxyl and carboxyl functional groups. Their composition suggests participating in many processes: electrostatic binding, donor-acceptor interaction and acting as nucleophilic and electrophilic agents. The aim of this work was to study the activity of these complexes as modifiers of α-Lrhamnosidases from Penicillium tardum, eupenicillium erubescens and Cryptococcus albidus.
The preparations of α-L-rhamnosidases were obtained from the culture supernatant of P. tardum [4], e. erubescens [5] and C. albidus [6], by filtration through 4 layers of gauze (for e. erubescens and P. tardum) or by centrifugation (for C. albidus), after separation of biomass and precipitation with ammonium sulfate to 90% saturation. The mixture was kept for 12-16 h at 4 °C and then centrifuged at 5000 g for 30 min. The precipitate was collected , dissolved in 1.5 volumes of 0.01 M phosphate buffer pH 7.0. A sample (about 20-30 mg of protein) was applied to a column (2.5×90 cm) with a neutral TSK-gel Toyopearl HW-60 (Toyo Soda, Japan) equilibrated with 0.01 M phosphate buffer, pH 7. 0. Fractions were eluted with the same buffer at a flow rate of 90 ml/h. The protein content was determined using SF-26 at 280 nm. Fractions containing α-Lrhamnosidase activity were collected, combined and concentrated (⁓ 5-fold) in vacuo. The obtained samples (4-5 ml, 7-10 mg of protein) were applied to Fractogel DEAE-650-s column (3×35 cm) (Merck, Germany) equilibrated with 0.01 M Tris-HCl buffer pH 7.8. Elution was performed with a linear NaCl gradient (0 -1 M, 200 ml) at a flow rate of 24 ml/h.
The α-L-rhamnosidase activity was determined by the Davis method [23] using naringin as a substrate. The specific α-L-rhamnosidase activity of the preparations was 120 units/mg of protein for e. erubescens, 27 units/mg of protein for P. tardum and 12 units/mg of protein for C. albidus (protein content -0.01 mg/ml).
When studying the effect of various germanium-containing compounds on the activity of enzymes, we used concentrations of 0.1 and 0.01% and time of exposure 0.5 h and 24 h. The studied compounds were dissolved in 0.1% DMSO.
All experiments were performed in 7 replicates. Student's t-test was used to perform statistical analysis. The data are presented as mean ± standard error (M ± m) and are considered significant at P < 0.05. The results presented in graphs were processed using Microsoft Excel 2007.

results and discussion
Previously, based on inhibitory and kinetic analysis with Dixon and Lineweaver-Burk plots and using group-specific reagents, we revealed some active functional groups in α-L-rhamnosidases from P. tardum, e. erubescens and C. albidus [7][8][9]. A significant inhibition (more than 50%) of the activity of all the studied enzymes by 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide methiodide may indicate an important role of carboxyl groups of aspartic or glutamine amino acids in the catalytic activity of the enzymes. However, partial inhibition may indicate the presence on the protein surface of other amino acid residues, which are also an important for the enzymatic reaction. A sulfhydryl group of cysteine is known to play important role in the activity of many proteins. Since p-chloromercuribenzoate, sodium arsenite, and N-ethylmaleimide inhibit the activity only of e. erubescens α-L-rhamnosidase, we suggest the presence of sulfhydryl groups in its active site. Moreover, this inhibition was not abolished by dithiothreitol, mercaptoethanol and cysteine. Involvement of the histidine's imidazole group in the activity of α-L-rhamnosidases of all three producers is suggested by a decrease in the enzymes activity by more than 90% when this group is modified by photooxidation in the presence of methylene blue and diethyl pyrocarbonate, which specifically reacts with histidine residues at pH 5.5-7.5. It was found that metal-containing groups do not involve in the catalysis by α-L-rhamnosidases of P. tardum and C. albidus, i.e., these enzymes are metal-independent enzymes, while the activity of the α-L-rhamnosidase of e. erubescens depends on the presence of metal ions (inhibition by EDTA, o-phenanthroline). Identification of catalytically important groups in the enzyme active site allows predicting its behavior in reaction media and control catalysis in order to optimize enzymatic processes in biotechnology.
It is known [1][2][3] that α-L-rhamnosidases belong to the enzymes, which do not require the presen ce of metals for their activity, however metal ions can change the conformation of the molecule and make active sites more accessible to the substrate, at that, the orientation of the distant groups can also be changed. It is not possible to predict precisely what effect each metal will have on enzyme activity.
Our research showed that the effect of germanium compounds on the activity of the studied α-L-rhamnosidases varied with different exposure times and concentrations. The most different effect was observed on the activity of α-L-rhamnosidase of P. tardum (Fig. 2, a, B). Thus, an increase in the activity of α-L-rhamnosidase of P. tardum up to 18% was observed at the action of compound (3) at a concentration 0.01% and exposure time 0.5 h (Fig. 2,  a), whereas an increase in exposure time to 24 h at the same concentration of compound (3) led to an increase in activity by 31% and at a concentration 0.1% -by 50%.
Compounds (7) and (9) at both concentrations and exposure time 0.5 h did not affect the activity of the studied α-L-rhamnosidase (activity was at the control level). All other compounds inhibited the activity of α-L-rhamnosidase from P. tardum. The most significant decrease (by 50-70%) in the activity was registered under the action of compound (4) at both concentrations.
The activity of the effectors decreased with increasing exposure time. Compound (5) was the only exception. Its effect (degree of inhibition) did not depend on exposure time and remained constant.
The maximum increase in the activity of α-Lrhamnosidase of e. erubescens occurred under the (2)
In the experiments with the enzyme from e. erubescens, along with compound (3), significant activity was also observed for compounds (6) and (8), which do not contain d-metal cations, but are able to bind to the active site of a metal-containing α-L-rhamnosidase. Differences in the influence of (1)-(11) on the activity of the enzymes from P. tardum, e. erubescens and C. albidus indicate that activation and inhibition occur with the involvement of different functional groups of these enzymes. Most likely, no bonds with such groups were formed in the case of C. albidus, hence most of the compounds were inactive towards it. The data presented differ from the previously reported results on citrate-germanate complexes as α-L-rhamnosidase modifiers [16] and indicate the essential role of the composition of complex germanium-containing anion, as the complex cations were the same.
It should be mentioned that when we have analyzed the publications on α-L-rhamnosidases, no other examples of metal-containing compounds capable of activating the enzyme were found, except for the ones we studied earlier [17][18][19]. The cations Cu 2+ , Ni 2+ , Fe 2+ , Zn 2+ in inorganic salts act as inhibitors of catalytic activity. That is, the unique combination of the biological properties of Ge, Cu, Ni, tartrate acid and 2,2′-bipyridine is a key factor. Therefore, the compounds [CuCl(bipy) 2 ] 2 [Ge 2 (OH) 2  Thus, coordination compounds with tartrategermanate anions and Cu (II) and Ni (II) 2,2′-bipyridine complex cations are currently of particular scientific and practical interest. The selected metal ions are essential elements and bonded to the biologically active ligands [10][11][12]. Related complexes demonstrate the ability to induce interferon and have immunostimulating effect, low toxicity, cardio-protective, antihypertensive, antiarrhythmic properties, and exert a beneficial effect in cardiovascular and chronic respiratory diseases, pneumonia, neuropsychiatric and metabolic disorders [21,22]. The compounds that can act as effectors on Penicillium tardum and eupenicillium erubescens α-Lrhamnosidases activity will have a wide range of practical applications.

Conflict of interest.
Authors have completed the Unified Conflicts of Interest form at http:// ukrbiochemjournal.org/wp-content/uploads/2018/12/ coi_disclosure.pdf and declare no conflict of interest.