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Respiratory nitrate reductase (Nar) from hydrocarbon-degrading bacterium Gordonia alkanivorans S7
New Biotechnology · Volume 25S · September 2009
dazine with the exception of weak activity with p-hydroquinone. Common catechol oxidase inhibitors (salicylhydroxamic acid and p-coumaric acid) inhibit the oxidase activity. Catechol oxidation activity was also detected in three other commercial catalases tested, from Aspergillus niger, human erythrocyte, and bovine liver, suggesting that this dual catalase-phenol oxidase activity may be a common feature of catalases.
ABSTRACTS
2.1.125 An Antarctic marine bacterium Pseudoalteromonas spp., strain 93, as a source of cold-adapted alkalistable subtilisin-like serine protease E. Gromek ∗ , M. Turkiewicz Technical University of Lodz, Lodz, Poland
doi:10.1016/j.nbt.2009.06.370
Gordonia alkanivorans S7 is an efficient degrader of fuel oil hydrocarbons that can simultaneously utilize oxygen and nitrate as electron acceptors. The nitrate respiratory pathway plays the crucial role in maintenance of its metabolic activity in polluted environments, where hydrophobic substrate considerably limits oxygen transfer to the cells. The high respiration activity, intensive growth and high degradation activity observed in the medium supplemented with nitrate indicated that G. alkanivorans S7 produced the respiratory nitrate reductase. Almost complete inhibition of the growth of this bacterium in presence of selected nitrate reductase inhibitors: sodium azide, sodium thiocyanate and sodium chlorate also provided evidence of Nar synthesis. A respiratory nitrate reductase (Nar) contained in the membrane fraction from this organism has been isolated using ion exchange chromatography on DEAE Biogel A and gel filtration on Sephacryl S-300 HR and preliminary characterized. Molecular-sieve chromatography on Sepharose CL 6-B revealed that molecular mass of the Nar was close to 443 kDa. Native PAGE of this reductase showed a single protein band corresponding to the similar molecular mass of 435 kDa. SDS-PAGE revealed that Nar consisted of three subunits of 103, 53 and 25 kDa. The enzyme was optimally active at pH 7.9 and 40◦ C. Km values for NO3 — (110 M) and for ClO3 — (138 M) were determined for a reduced viologen as an electron donor. The purified Nar did not use NADH as the electron donor to reduce nitrate or chlorate. Azide was a strong inhibitor of its activity. Our results imply that enzyme isolated from G. alkanivorans S7 is a respiratory membrane-bound nitrate reductase. This is the first report of purification of a nitrate reductase from Gordonia species. Our findings support the use of nitrate in mixed electron acceptor conditions as a feasible strategy for bioremediation contaminated aquifers and soil.
Biotechnological applications of extremophiles and their enzymes, called extremozymes, have been the driving force in both academic and industrial research of those microorganisms. The diversity of environments to which extremophiles and their biomolecules have adapted offers unique opportunity for their applications under harsh conditions in various branches of industry making technological processes more environmentally friendly. Despite the fact that the psychrosphere accounts for more than 80% of biosphere and thus psychrophiles and also their enzymes are more wide spread in nature, more studies have been focused on thermozymes than on cold-adapted biocatalysts. The marine bacterium Pseudoalteromonas spp., strain 93, from the pure culture collection at the Institute of Technical Biochemistry of the Technical University of Lodz, isolated from alimentary tract of Antarctic krill Euphausia superba Dana secretes alkalistable subtilisin-like serine protease. The purified protease has a molecular mass of approximately 45 kDa and is stabilized by Ca2+ ions. The enzyme is optimally active at 45◦ C and pH 10.0 against natural and synthetic substrates and retains 12—30% maximum activity in the temperature range between 0 and 20◦ C. The protease is stable up to 55◦ C for 60 min, and at pH of 11.5 it displays 70% maximum activity. The protein exhibits satisfactory stability in NaCl and urea solutions. During 24 hours incubation at 4◦ C it lost about 30% and 40% of an initial activity in 5 M NaCl and 8 M urea, respectively. After 60 min incubation at 4◦ C in 1% solution of SDS the protease activity was decreased by about 40%. The efficient hydrolysis of N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (N-sucAAPF-pNA) and N-benzoyl-L-tyrosine ethyl ester (BzTyrOEt) by this enzyme and its complete inhibition by phenylmethylsulfonyl fluoride (PMSF) suggest that it is a subtilisin-like serine protease. The enzyme had a Km of 2.49 mM for N-SuccAAPFpNA at 30◦ C and the value of the activation energy in N-SuccAAPFpNA hydrolysis was found to be 40.7 kJ × mol−1 in the temperature range of 15—45 ◦ C. The properties of serine protease from Antarctic marine bacterium Pseudoalteromonas spp., strain 93, such as alkaline optimal pH, alkalistability and relatively high activity in cold, imply its potential usefulness for manufacturing of detergents to be applied at low temperatures. It can also be used in biocatalysis in organic solvents.
doi:10.1016/j.nbt.2009.06.371
doi:10.1016/j.nbt.2009.06.372
2.1.124 Respiratory nitrate reductase (Nar) from hydrocarbondegrading bacterium Gordonia alkanivorans S7 I. Romanowska ∗ , E. Kwapisz, M. Mitka, S. Bielecki Institute of Technical Biochemistry, Technical University of Lodz, Lodz, Poland
www.elsevier.com/locate/nbt S93
dazine with the exception of weak activity with p-hydroquinone. Common catechol oxidase inhibitors (salicylhydroxamic acid and p-coumaric acid) inhibit the oxidase activity. Catechol oxidation activity was also detected in three other commercial catalases tested, from Aspergillus niger, human erythrocyte, and bovine liver, suggesting that this dual catalase-phenol oxidase activity may be a common feature of catalases.
ABSTRACTS
2.1.125 An Antarctic marine bacterium Pseudoalteromonas spp., strain 93, as a source of cold-adapted alkalistable subtilisin-like serine protease E. Gromek ∗ , M. Turkiewicz Technical University of Lodz, Lodz, Poland
doi:10.1016/j.nbt.2009.06.370
Gordonia alkanivorans S7 is an efficient degrader of fuel oil hydrocarbons that can simultaneously utilize oxygen and nitrate as electron acceptors. The nitrate respiratory pathway plays the crucial role in maintenance of its metabolic activity in polluted environments, where hydrophobic substrate considerably limits oxygen transfer to the cells. The high respiration activity, intensive growth and high degradation activity observed in the medium supplemented with nitrate indicated that G. alkanivorans S7 produced the respiratory nitrate reductase. Almost complete inhibition of the growth of this bacterium in presence of selected nitrate reductase inhibitors: sodium azide, sodium thiocyanate and sodium chlorate also provided evidence of Nar synthesis. A respiratory nitrate reductase (Nar) contained in the membrane fraction from this organism has been isolated using ion exchange chromatography on DEAE Biogel A and gel filtration on Sephacryl S-300 HR and preliminary characterized. Molecular-sieve chromatography on Sepharose CL 6-B revealed that molecular mass of the Nar was close to 443 kDa. Native PAGE of this reductase showed a single protein band corresponding to the similar molecular mass of 435 kDa. SDS-PAGE revealed that Nar consisted of three subunits of 103, 53 and 25 kDa. The enzyme was optimally active at pH 7.9 and 40◦ C. Km values for NO3 — (110 M) and for ClO3 — (138 M) were determined for a reduced viologen as an electron donor. The purified Nar did not use NADH as the electron donor to reduce nitrate or chlorate. Azide was a strong inhibitor of its activity. Our results imply that enzyme isolated from G. alkanivorans S7 is a respiratory membrane-bound nitrate reductase. This is the first report of purification of a nitrate reductase from Gordonia species. Our findings support the use of nitrate in mixed electron acceptor conditions as a feasible strategy for bioremediation contaminated aquifers and soil.
Biotechnological applications of extremophiles and their enzymes, called extremozymes, have been the driving force in both academic and industrial research of those microorganisms. The diversity of environments to which extremophiles and their biomolecules have adapted offers unique opportunity for their applications under harsh conditions in various branches of industry making technological processes more environmentally friendly. Despite the fact that the psychrosphere accounts for more than 80% of biosphere and thus psychrophiles and also their enzymes are more wide spread in nature, more studies have been focused on thermozymes than on cold-adapted biocatalysts. The marine bacterium Pseudoalteromonas spp., strain 93, from the pure culture collection at the Institute of Technical Biochemistry of the Technical University of Lodz, isolated from alimentary tract of Antarctic krill Euphausia superba Dana secretes alkalistable subtilisin-like serine protease. The purified protease has a molecular mass of approximately 45 kDa and is stabilized by Ca2+ ions. The enzyme is optimally active at 45◦ C and pH 10.0 against natural and synthetic substrates and retains 12—30% maximum activity in the temperature range between 0 and 20◦ C. The protease is stable up to 55◦ C for 60 min, and at pH of 11.5 it displays 70% maximum activity. The protein exhibits satisfactory stability in NaCl and urea solutions. During 24 hours incubation at 4◦ C it lost about 30% and 40% of an initial activity in 5 M NaCl and 8 M urea, respectively. After 60 min incubation at 4◦ C in 1% solution of SDS the protease activity was decreased by about 40%. The efficient hydrolysis of N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (N-sucAAPF-pNA) and N-benzoyl-L-tyrosine ethyl ester (BzTyrOEt) by this enzyme and its complete inhibition by phenylmethylsulfonyl fluoride (PMSF) suggest that it is a subtilisin-like serine protease. The enzyme had a Km of 2.49 mM for N-SuccAAPFpNA at 30◦ C and the value of the activation energy in N-SuccAAPFpNA hydrolysis was found to be 40.7 kJ × mol−1 in the temperature range of 15—45 ◦ C. The properties of serine protease from Antarctic marine bacterium Pseudoalteromonas spp., strain 93, such as alkaline optimal pH, alkalistability and relatively high activity in cold, imply its potential usefulness for manufacturing of detergents to be applied at low temperatures. It can also be used in biocatalysis in organic solvents.
doi:10.1016/j.nbt.2009.06.371
doi:10.1016/j.nbt.2009.06.372
2.1.124 Respiratory nitrate reductase (Nar) from hydrocarbondegrading bacterium Gordonia alkanivorans S7 I. Romanowska ∗ , E. Kwapisz, M. Mitka, S. Bielecki Institute of Technical Biochemistry, Technical University of Lodz, Lodz, Poland
www.elsevier.com/locate/nbt S93