Influence of New Types of Biscitratogermanates on Penicillium restrictum α-L-Rhamnosidase

Abstract

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 α-L-rhamnosidases. Their wide use to solve current biological-medical and chemical-technological problems stimulates researchers to search for compounds capable of influencing their catalytic activity. Therefore, the purpose of this work was to isolate and purify α-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. The object of the study was α-L-rhamnosidase of P. restrictum. Its purification was carried out by gel filtration and ion exchange chromatography on TSK-gels and Sepharose 6B. The activity of α-L-rhamnosidase was determined using the Davis method with naringin as a substrate. As modifiers of enzyme activity, purposefully synthesized multiligand germanium-3d-metal complexes with citric acid, phenanthroline, and bipyridine ([Ni(bipy)₃][Ge(HCit)₂]·3H₂O (1); [Ni(phen)₃][Ge(HCit)₂]·2H₂O (2); [{Cu(bipy)₂}₂Ge(m-Cit)₂]·12Н₂О (3); [{Cu(phen)₂}₂Ge(m-Cit)₂]·13H₂O (4); [Zn(bipy)₃][Ge(HCit)₂]·2H₂O (5); [Zn(phen)₃][Ge(HCit)₂]·3H₂O (6)), were used. Results. From the supernatant of culture fluid of P. restrictum, α-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 α-L-rhamnosidase of P. restrictum. All of them manifest themselves either as activators 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 affected by the time of exposure to the compounds: at a concentration of 0.01% for 1h, 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 α-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.