“…In order to confirm the identity of the CAT enzyme, the 62.7-kDa band was excised from SDS-PAGE and subjected to MALDI TOF/TOF analysis. The experimentally obtained masses were compared with the theoretical peptide masses of proteins stored in the NCBInr database using the mass search program Mascot as previously described [37]. The result of the peptide mass fingerprinting showed that the enzyme matched with the information reported for the CAT from Serratia proteamaculans 568.…”
Section: Resultsmentioning
confidence: 99%
“…Catalase enzyme was routinely purified from I1P cells at 22 • C. For the preparation of the crude extract, 20 g of cells were lysed with a modified cellular disruption method [37]. Briefly, cells were resuspended in 80 mL of 50 mM Tris HCl buffer (pH 7.5) containing 1 mM EDTA and lysozyme (1 mg•mL −1 ) and incubated at 37 • C for 1 h. Then, the sample was disrupted using a sonicator (Branson sonifier 450, Branson, Danbury, CT, USA) and the cell debris was removed by centrifugation (9000× g for 30 min).…”
Section: Enzyme Purificationmentioning
confidence: 99%
“…For determination of CAT thermostability, the enzyme was placed in small tubes with O-ring-sealed caps and incubated at 50 • C in a dry bath (MD-02N-220, Major Science, Saratoga, CA, USA) for 0 to 6 h, as previously described [37]. Samples were taken at different times and assayed for enzyme activity.…”
Microorganisms present in Antarctica have to deal not only with cold temperatures but also with other environmental conditions, such as high UV radiation, that trigger the generation of reactive oxygen species. Therefore, Antarctic microorganisms must have an important antioxidant defense system to prevent oxidative damage. One of these defenses are antioxidant enzymes, such as catalase, which is involved in the detoxification of hydrogen peroxide produced under oxidative conditions. Here, we reported the isolation and partial characterization of an Antarctic bacterium belonging to the Serratia genus that was resistant to UV-C radiation and well-adapted to cold temperatures. This microorganism, denominated strain I1P, was efficient at decreasing reactive oxygen species levels produced after UV-C irradiation. Genomic and activity assays suggested that the enzymatic antioxidant defense mechanisms of strain I1P, especially its catalase enzyme, may confer UV resistance. This catalase was active in a wide range of temperatures (20–70 °C), showing optimal activity at 50 °C (at pH 7.0), a remarkable finding considering its psychrotolerant origin. In addition, this enzyme was thermostable, retaining around 60% of its activity after 6 h of incubation at 50 °C. The antioxidant defense systems of strain I1P, including its surprisingly thermoactive and thermostable catalase enzyme, make this microorganism a good source of biocompounds with potential biotechnological applications.
“…In order to confirm the identity of the CAT enzyme, the 62.7-kDa band was excised from SDS-PAGE and subjected to MALDI TOF/TOF analysis. The experimentally obtained masses were compared with the theoretical peptide masses of proteins stored in the NCBInr database using the mass search program Mascot as previously described [37]. The result of the peptide mass fingerprinting showed that the enzyme matched with the information reported for the CAT from Serratia proteamaculans 568.…”
Section: Resultsmentioning
confidence: 99%
“…Catalase enzyme was routinely purified from I1P cells at 22 • C. For the preparation of the crude extract, 20 g of cells were lysed with a modified cellular disruption method [37]. Briefly, cells were resuspended in 80 mL of 50 mM Tris HCl buffer (pH 7.5) containing 1 mM EDTA and lysozyme (1 mg•mL −1 ) and incubated at 37 • C for 1 h. Then, the sample was disrupted using a sonicator (Branson sonifier 450, Branson, Danbury, CT, USA) and the cell debris was removed by centrifugation (9000× g for 30 min).…”
Section: Enzyme Purificationmentioning
confidence: 99%
“…For determination of CAT thermostability, the enzyme was placed in small tubes with O-ring-sealed caps and incubated at 50 • C in a dry bath (MD-02N-220, Major Science, Saratoga, CA, USA) for 0 to 6 h, as previously described [37]. Samples were taken at different times and assayed for enzyme activity.…”
Microorganisms present in Antarctica have to deal not only with cold temperatures but also with other environmental conditions, such as high UV radiation, that trigger the generation of reactive oxygen species. Therefore, Antarctic microorganisms must have an important antioxidant defense system to prevent oxidative damage. One of these defenses are antioxidant enzymes, such as catalase, which is involved in the detoxification of hydrogen peroxide produced under oxidative conditions. Here, we reported the isolation and partial characterization of an Antarctic bacterium belonging to the Serratia genus that was resistant to UV-C radiation and well-adapted to cold temperatures. This microorganism, denominated strain I1P, was efficient at decreasing reactive oxygen species levels produced after UV-C irradiation. Genomic and activity assays suggested that the enzymatic antioxidant defense mechanisms of strain I1P, especially its catalase enzyme, may confer UV resistance. This catalase was active in a wide range of temperatures (20–70 °C), showing optimal activity at 50 °C (at pH 7.0), a remarkable finding considering its psychrotolerant origin. In addition, this enzyme was thermostable, retaining around 60% of its activity after 6 h of incubation at 50 °C. The antioxidant defense systems of strain I1P, including its surprisingly thermoactive and thermostable catalase enzyme, make this microorganism a good source of biocompounds with potential biotechnological applications.
“…We focused our interest in GDH activity due to its promising biotechnological applications. We choose for the measurement of activity the direction of oxidative deamination reaction and NAD + as coenzyme due to its higher thermal stability compared with NADP + [ 26 ]. GDH activity was measured in PID15 isolate at two different temperatures 37 and 50 °C (Fig.…”
Section: Resultsmentioning
confidence: 99%
“…The specific activity was determined for the oxidative deamination reaction at 37 °C and 50 °C. As control glutamate dehydrogenase from GWE1 was used [ 26 ]. Error bars show the variation obtained from three biological replicates Fig.…”
BackgroundThe Antarctic continent is a source of extreme microorganisms. Millions of years of isolation have produced unique biodiversity with adaptive responses to its extreme environment. Although the Antarctic climate is mainly cold, the presence of several geothermal sites, including thermal springs, fumaroles, hot soils and hydrothermal vents, provides ideal environments for the development of thermophilic and hyperthermophilic microorganisms. Their enzymes, called thermoenzymes, are the focus of interest in both academic and industrial research, mainly due to their high thermal activity and stability. Glutamate dehydrogenase, is an enzyme that plays a key role in the metabolism of carbon and nitrogen catalyzing reversibly the oxidative deamination of glutamate to alpha-ketoglutarate and ammonium. It belongs to the family of oxidoreductases, is widely distributed and it has been highly regarded for use as biosensors, particularly for their specificity and ability to operate in photochemical and electrochemical systems. However, the use of enzymes as biosensors is relatively problematic due to their instability to high temperatures, organic solvents and denaturing agents. The purpose of this study is to present the partial characterization of a thermophilic microorganism isolated from Deception Island, Antarctica, that displays glutamate dehydrogenase activity.ResultsIn this work, we report the isolation of a thermophilic microorganism called PID15 from samples of Deception Island collected during the Antarctic Scientific Expedition ECA 46. This microorganism is a thermophile that grows optimally at 50 °C and pH 8.0. Scanning electron microscopy shows rod cells of 2.0 to 8.0 µm of length. Phylogenetic analysis of 16S rRNA gene revealed that this microorganism is closely related to Bacillus gelatini. This microorganism contains a thermostable glutamate dehydrogenase with optimal activity at pH 8.0 and temperatures for its activity from 37 to 50 °C, range of temperature of interest for biotechnological applications. This glutamate dehydrogenase is a highly thermostable enzyme.ConclusionThis is the first report of a microorganism from Antarctica containing a thermostable glutamate dehydrogenase that maintains its activity in a broad range of temperatures making it of potential interest for biotechnological applications.
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