Naringinase which was extracted from the fermented broth of Cryptococcus albidus was purified about 42-folds with yield 0.7% by sulfate fractionation and chromatography on Toyopearl HW-60, Fractogel DEAE-650-s, and Sepharose 6B columns. Molecular weight of protein determined by gel filtration and SDS-PAGE was 50 kDa. Naringinase of C. albidus includes high content of the dicarbonic and hydrophobic amino acids. Enzyme contains also carbohydrate component, represented by mannose, galactose, rhamnose, ribose, arabinose, xylose, and glucose. The enzyme was optimally active at pH 5.0 and 60 °C. Naringinase was found to exhibit specificity towards p-nitrophenyl-α-L-rhamnose, p-nitrophenyl-β-D-glucose, naringin, and neohesperidin. Its K towards naringin was 0.77 mM and the V was 36 U/mg. Naringinase was inhibited by high concentrations of reaction product-L-rhamnose. Enzyme revealed stability to 20% ethanol and 500 mM glucose in the reaction mixture that makes it possible to forecast its practical use in the food industry in the production of juices and wines.

Despite the fact that in recent years there has been a certain enhancing interest in the study of marine microorganisms, nevertheless, marine bacteria as producers of biologically active substances, in particular enzymes, are still poorly studied. The marine biota is significantly different from the terrestrial one; therefore, there is a high probability of detecting in the marine environment different from terrestrial bacteria producers of enzymes with unique specificity and activity, for the needs of modern biotechnology. Proteolytic enzymes play an important role in these studies. Since the majority of microbial producers are characterized by a number of serious deficiencies, in particular, most of the elastase producers described in the literature are pathogenic for humans, the search for new, effective producers continues to be an urgent problem, given that highly active producers of proteolytic enzymes, in particular elastase, are generally absent in Ukraine. In this regard, the purpose of this work was to screen microorganisms isolated from the Black Sea for the presence of effective producers of proteolytic enzymes. Methods. We used methods of determining proteolytic (caseinilytic, elastolytic, fibrinolytic, fibrinogenolytic) activity. Results. The study of the enzymatic activity of the isolates showed that on the 10th day of cultivation in the supernatant of the culture liquid, caseinolytic activity was not detected only in one isolate 56, whereas very insignificant activity was observed in isolates 7, 20, and 50. The maximum activity was detected in isolate 247 (0.2 units/mL), and lower one - in isolates 46 (0.16 U/mL), 52 (0.15 U/mL), 51 (0.135 U/mL), 54 (0.08 U/mL), and 44 (0.05 U/mL). Of the 10 studied isolates, elastase activity was found only in four of them. The highest activity was found in isolates 51 and 54 (20.83 and 19.96 U/mL, respectively). Lower levels of activity (15.62 U/mL and 12.15 U/mL, respectively) were shown by isolates 52 and 247. The studied isolates also differed in their ability to hydrolyze fibrin and fibrinogen. T e highest fi brinolytic activity (2.33 U/mL) was found in isolates 46 and 54, significantly lower in isolate 20 (0.5 U/mL) and isolate 44 (0.33 U/mL). The rest isolates did not show fibrinolytic activity. As for fibrinogenolytic activity, it was noted in 6 studied cultures. The highest levels of activity were observed in isolate 51 (1.16 U/mL). Lower activity was found in isolates 54 (0.66 U/mL), 7 (0.5 U/mL), and 247 (0.33 U/mL). In isolate 50, it was minimal (0.083 U/mL). Conclusions. No correlation was found between elastase, fibrinolytic and fibrinogenic activity in the studied isolates. Thus, isolates 51, 54 and, to a lesser extent, 52 and 247 synthesize elastase activity. The highest fibrinolytic activity was in isolates 46 and 54, and fibrinogenolytic activity was in isolate 51. It was shown that the Black Sea is rich in marine bacterial species, which can be effective producers of a number of practically important enzymes, in particular, proteolytic ones with specificity to elastin, fibrin, and fibrinogen, which can be promising for implementation in biotechnological processes.

Вивчення впливу іонів металів, аніонів та специфічних хімічних реагентів на активність α-L-рамнозидази Penicillium tardum. Методи. a-L-Рамнозидазну активність визначали за швидкістю гідролізу n-нітрофеніл-a-L-рамнопіранозиду. В дослідах щодо впливу на активність α-L-рамнозидази використовували іони металів у вигляді сульфатів, лише Ag +-у вигляді нітрату, аніони-у вигляді солей калію або натрію, а також наступні специфічні хімічні реагенти: етилендіамінтетраацетат, о-фенантролін, дитіотреїтол, L-цистеїн, β-меркаптоетанол, п-хлормеркурібензоат, N-етилмалеїмід, 1-[3-(диметиламіно)пропіл]-3-етилкарбодимід метіодид. Всі речовини досліджували в концентрації 10-3 М. Результати. Cуттєвий вплив на α-L-рамнозидазу P. tardum мали іони Ag + та Cd 2+ (в кінцевій концентрації 10-3 М), які повністю інгібували активність ензиму, тоді як Ca 2+ підвищував активність досліджуваної α-L-рамнозидази P. tardum на 60%. При вивченні дії різних аніонів встановлено, що сульфіт на 73% інгібує активність ферменту, в той час як аніони AsO 3-2 та CO 3-2 активують α-L-рамнозидазу P. tardum на 100 і 75% відповідно. Висновки. В каталізі, що здійснюється α-L-рамнозидазою P. tardum, не беруть участі групи, які містять атоми металів. Присутність іонів Ag + , Cd 2+ в системі інгібує швидкість ензимної реакції, тоді як іони Ca 2+ підвищують активність досліджуваної α-L-рамнозидази P. tardum на 60%. Оскільки 1-[3-(диметиламіно) пропіл]-3-етилкарбодимід метіодид інгібує активність α-L-рамнозидази на 50-73%, можна припустити, що в молекулі α-L-рамнозидази присутні функціонально активні карбоксильні групи. Ключові слова: α-L-рамнозидаза, Penicillium tardum, іони металів, специфічні хіміч ні реа генти.

No abstract

In the present study authors establish the influence of complex cation on the structure formation, crystal packing, antimicrobial activity and enzyme activation of the previously described isostructural [M(phen)3]2[Sn2(μ‐Tart)2(H2Tart)2] complexes (M(II)=Fe(1), Co(2), Ni(3), Cu(4), Zn(5)). Despite the similarity of composition and structure, compounds 1–5 showed different level of antimicrobial activity. A certain pattern in their effectiveness – decreasing of minimal concentrations, resembles the Irving‐Williams series (Fe(II) < Co(II) < Ni(II) < Cu(II) > Zn(II)) and, according to the crystal field theory, correlates with the thermodynamic stability of octahedral cations [M(phen)3]2+. Quantum chemical calculations of cations revealed the insights of the “cations‐cell membranes” interactions and allowed authors to conclude that the cations in compounds 4 and 5 are the most lipophilic, have the highest affinity to the lipophilic cell membrane and, as a result, are more bioactive. It was established that compounds 1–5 had a different influence on the activity of studied α‐L‐rhamnosidases Penicillium tardum ІMB F‐100074 and Penicillium restrictum ІMB F‐100139. Correlation of these results with the crystal voids calculations and Hirshfeld surface analysis allow authors to suggest that presence of the bigger number of water molecules, generation of the branched network of hydrogen bonds and formation of active intermediate complex “enzyme – effector – substrate”, leads to the biggest intensification of activation properties for the compounds 2 and 5.