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The aim of the work was to determine the influence of a number of coordination compounds of germanium, in particular cation-anionic type, on the activity of α-L-rhamnosidases of three producers: Cryptococcus albidus, Eupenicillium erubescens and Penicillium tardum ІМВ F-100074. Activity of α-L-rhamnosidases was determined according to Davis method, using naringin as substrate. The specific α-L-rhamnosidase activity of preparations was 12 units/mg of protein for C. аlbidus, 120 units/mg for E. еrubescens and 27 units/mg for P. tardum. Protein concentration in preparations was 0.01 mg/ml. Gernanium compounds were synthesized in alcohol-water solution according to standardized methods. In all compounds, the same bis (citrato) germanate anion is realized; however, the composition of the cation changes: the protonated form of phenanthroline and bipyridine or the complex cations of d-metals. It was established that none of the complex compounds exhibited an inhibitory effect on the activity of the enzymes under study. Maximum activating effect was observed with compound tris(bi-pyridine)nickel(II) bis(citrate)germanate monohydrate in concentration of 0.1% on α-L-rhamnosidases of C. albidus (10-fold), E. еrubescens (2.5-fold) and P. tardum (5-fold). Notably, enzymatic activity increased by 45, 47 and 60% respectively in presences of compound bi-pyridine bis(citrate)germanate dihydrate in same concentration. Hence, it was shown that these two compounds can be used as effectors of studied enzymes.

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]