Bacteria of the Black Sea are Producers of α-L-Rhamnosidase

Abstract

The search for new producers of α-L-rhamnosidases and the study of their properties are constantly carried out, which makes it possible to identify enzymes with unique properties. Thus, the α-L-rhamnosidases producers of marine species of microorganisms can radically differ in habitat conditions from terrestrial ones. Previously, we have isolated the producers of α-L-rhamnosidases from a number of representatives of the Black Sea microbiota. However, the results of these studies did not allow us to isolate a strain promising for further study of the α-L-rhamnosidase synthesized by it. Therefore, the purpose of this work was to further search for effective producers of α-L-rhamnosidases among the microbiota of the Black Sea obtained from its different depths. Methods. Glycosidase activities were determined by the Romero and Davis methods. Results. The study of α-L-rhamnosidase activity in the dynamics of growth of 10 cultures isolated from the Black Sea showed that the only glycosidase activity, which was found on the third day of cultivation in six (07, 044, 050, 052, 054, 247) of ten cultures studied, was toward α-L-rhamnosidase. However, on the 5th day of cultivation, an increase (and in some strains, the appearance) of activity was noted in all tested cultures (from 0.01 to 0.12 U/mL). On the 7th day of cultivation, α-L-rhamnosidase activity in the supernatant of the culture liquid ranged from 0.02 to 0.2 U/mL. The highest activity (0.2 U/mL) was found in strain 052. On the 10th day of cultivation, the maximum activity (0.55 U/mL) was noted in culture 052 and slightly lower (0.35 U/mL, 0.28 U/mL, and 0.23 U/mL) in cultures 044, 050, and 054 respectively. Cultures 051, 020, and 247, which showed the same activity (0.1 U/mL), as well as 056 (0.09 U/mL) were an order of magnitude less active. The minimum activity was noted in culture 046 (0.03 U/mL). In the supernatant of the culture liquid of strain 07 on the 10th day of cultivation, α-L-rhamnosidase was absent at all. Since the highest α-L-rhamnosidase activity was found in the supernatants of culture liquids of 5 strains (044, 052, 054, 056, and 247), partially purified complex preparations of those α-L-rhamnosidases were obtained for further research. The study of the substrate specificity of complex enzyme preparations of α-L-rhamnosidases of strains 044, 051, 052, 056, and 247 on natural flavonoids, such as naringin, neohesperidin, and rutin, indicated that α-L-rhamnosidase obtained from strain 052 showed the highest activity on three investigated substrates: rutin, naringin (0.55 U/mL), and neohesperidin (0.52 U/mL). In addition to natural substrates, complex preparations of α-L-rhamnosidases of strains 052, 054, 056, and 247 also hydrolyzed synthetic derivatives of monosaccharides, such as p-nitrophenyl-α-L-rhamnopyranoside and p-nitrophenyl-β-D-glucopyranoside. The maximum activity (0.15 U/mL) was noted in strain 052, whereas strain 044 was unable to hydrolyze synthetic substrates. Conclusions. Complex enzyme preparations of α-L-rhamnosidase obtained from strain 052 are promising for further investigations. They showed the highest activity both on three natural substrates, such as rutin, naringin, and neohesperidin, as well as on such synthetic derivatives of monosaccharides as p-nitrophenyl-α-L-rhamnopyranoside and p-nitrophenyl-β-D-glucopyranoside.