Marine Actinobacteria – Producers of Enzymes with α-L-Rhamnosidase

Self Cite

About 2 million tons of feathers are produced annually around the world as a by-product of poultry farming. Due to the lack of funds and the complexity of processing, they have become one of the main environmental pollutants. The biodegradation of feathers by keratinolytic microorganisms has proven to be an effective, environmentally friendly and cost-effective method of bioconverting feather waste into a nutritious, balanced and easily digestible product that contains free amino acids, peptides and ammonium ions. Aim. To investigate the ability of marine actinobacteria to synthesize enzymes with keratinolytic activity and to study some of the physicochemical properties of the most active enzyme preparation. The object of the study was 10 strains of actinobacteria isolated from bottom sediments in the area of the Pradneprovsky trench of the Black Sea shelf. Methods. Caseinolytic (general proteolytic) activity was determined by the Anson method modified by Petrova, based on the quantitative determination of tyrosine, which is formed during the enzymatic hydrolysis of casein. Keratinase activity was determined by UV absorption at 280 nm of the hydrolysis products of keratin-containing raw materials. The cultivation of actinobacteria was carried out in a liquid nutrient medium with the addition of defatted chicken feathers as the main source of carbon and nitrogen. Results. The ability to hydrolyze keratin was found in five cultures. Moreover, all the strains studied were practically unable to break down casein. The Acty 9 strain (12 U/ml) showed the highest keratinase activity. Additional introduction of NaCl to the nutrient medium did not have a positive effect on the enzymes synthesis. The study of the physicochemical properties of the enzyme preparation Acty 9 showed that the pH and thermooptimum were 9.0 and 60°C, respectively. It retained 100% of the initial activity in the range of pH 7.0–10.0 after 3 h and 95% activity at pH 8.0 after 24 h of incubation. The studied enzyme preparation was thermostable, since it remained active for 3 h at 50°C and 1 h at 60°C. Conclusions. The extracellular keratinase synthesized by actinobacterium Acty 9 is promising for further research, since the enzyme is pH and thermostable and is not inferior in its physicochemical properties to those previously described in the literature.

Section: Methodsmentioning

confidence: 90%

Screening of Enzyme Producers with Keratinase Activity among Marine Actinobacteria

Avdiyuk¹,

Ivanytsia²,

Varbanets³

2021

Self Cite

“…Th e reaction mixture consisting of 10 mg of cut small defatted chicken feathers, 2.5 mL of 0.05 M boron-borate buff er (pH 9.0), and 1 mL of CLS was kept in a thermostat at 37 °C for 3 hr and then fi ltered. To determine KerA, two controls were used: 10 mg of the feathers, 2.5 mL of 0.05 M boron-borate buff er (pH 9.2), and 1 mL of distilled water (1); 2.5 mL of 0.05 M boron-borate buff er (pH 9.2) and 1 mL of CLS (2). Th e sum of the two controls was subtracted from the A 280 values obtained by measuring the optical density of fi ltrates.…”

Section: Methodsmentioning

confidence: 99%

“…Marine microorganisms sequester large amounts of carbon and produce much of the world's oxygen. For a long time, the main interest in the marine environment, considered extreme, was focused on the isolation and identifi cation of natural products with biological properties, and for that, numerous organisms and chemical structures were studied [1,2]. Marine bacteria isolated from various substrates, such as sediments, seawater, and mangrove detritus, are producers of enzymes with diff erent activities, including amylase, cellulase, alginate lyase, chitinase, glucosidase, inulinase, keratinase, ligninase, xylanase, and others [3].…”

mentioning

confidence: 99%

Untitled

2023

For a long time, the main interest in the marine environment, considered extreme, was the isolation and identifi cation of natural products with biological properties, and for that, numerous organisms and chemical structures have been studied. Th us, marine bacteria isolated from various substrates, such as sediments, seawater, and mangrove detritus, are producers of enzymes with diff erent activities, i.e., amylase, cellulase, alginate lyase, chitinase, glucosidase, inulinase, keratinase, ligninase, xylanase, and others. Nowadays, researchers are also focusing on the enzymes produced in the marine environment that can present special properties. Th erefore, the aim of this study was to investigate the ability of marine strains of microorganisms to exhibit cellulase, β-mannanase, keratinase, and caseinolytic activities. Methods. Enzymatic activities were studied in the culture liquid supernatant. To determine β-mannanase and cellulase activities, guar gum galactomannan and Na-carboxymethylcellulose respectively were used as substrates. Casein and crushed defatted feathers served as substrates for the determination of proteolytic activity. Results. Growing 10 cultures of microorganisms on a nutrient medium containing chicken feathers as the sole source of carbon and nitrogen (nutrient medium 1) did not give positive results. When using medium 2, active growth was observed in four of the studied strains (51, 52, 54, 247) in the supernatant of culture liquid (CLS), the activity of which both to keratin (6.0-16.0 U/mL) and casein (0.025-0.33 U/mL) was found. In the CLS of only six of the 10 studied cultures (7, 20, 51, 52, 50, 247), cellulase and β-mannanase activities were observed. Th e highest cellulase activity was found in culture 20 (1.8 U/mL). Th e activity of culture 7 was somewhat lower (1.0 U/mL). An insignifi cant activity was noted in cultures 54 (0.06 U/mL), 56, and 50 (0.05 U/mL). Trace levels of activity were observed in culture 247. Conclusions. Strains 7, 20, 247, and 51, for the fi rst time isolated from the Black Sea, are promising for further studies as producers of cellulase, β-mannanase, keratinase, and caseinolytic enzymes.

“…Th e high biotechnological potential of marine microorganisms has recently stimulated the active screening of glycosidases among marine bacteria [9][10][11][12][13]. Previously, we have shown [5] that α-Lrhamnosidase of a representative of actinobacteria isolated from the Black Sea, namely strain Acty 5, is more active toward such natural substrates as rutin, naringin, and neohesperidin than toward synthetic substrates. Similar specifi city was also found in other bacterial α-L-rhamnosidases [1][2][3].…”

Section: Conclusion Complex Enzyme Preparations Of α-L-rhamnosidase O...mentioning

confidence: 99%

“…Th ese may be α-L-rhamnosidases, the producers of which are marine species of microorganisms, radically different in habitat conditions from the terrestrial ones. Previously [4,5], 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 α-L-rhamnosidase synthesized by it.…”

mentioning

confidence: 99%

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

Гудзенко¹,

Ivanytsia²,

Varbanets³

2023

Self Cite

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.