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Purification, characterization and identification of a novel bifunctional catalase-phenol oxidase from Scytalidium thermophilum
New Biotechnology · Volume 25S · September 2009
ABSTRACTS
lases reported have ability to hydrolyze high concentration raw starch mashes effectively because of the inhibitory effect of the substrates or products on the enzyme activity. In the present study, we report the production, purification, characterization and partial amino acid sequences of a raw starch digesting ␣-amylase produced by Anoxybacillus amylolyticus isolated from Mount Rittmann in Antarctica.
and the polymer contained 88.1 and 12.8 mol% 3HB and 3HV monomer, respectively.
doi:10.1016/j.nbt.2009.06.368
Purification, characterization and identification of a novel bifunctional catalase-phenol oxidase from Scytalidium thermophilum
2.1.122
D. Sutay Kocabas 1,∗ , U. Bakir 1 , S.E. Phillips 2 , M.J. Mcpherson 2 , Z.B. Ogel 3
Production of poly(3-hydroxyalkanoic acids) by moderately halophilic bacteria isolated from solar salterns A.K. Paul 1,∗ , A. Biswas 2 1
2
Department of Botany, University of Calcutta, Kolkata, India University of Calcutta, Kolkata, India
Moderately halophilic bacteria capable of growing optimally at 0.5—2.5 M NaCl have long been attracted the attention of microbiologists as sources of biopolymers, more specifically the biopolyesters, poly(3-hydroxyalkanoic acid) [P(3HB)] and its copolymer, poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) [P(3HB-co-3HV)]. These polyesters represent the most extensively studied members of poly(hydroxyalkanoic acids) [PHAs] and are known to posses properties similar to petrochemical based plastics. In addition, these PHA plastics are completely biodegradable in the environment, biocompatible and could be produced from renewable sources. The present investigation is aimed at to explore the moderately halophilic heterotrophic bacteria from hypersaline environments of coastal West Bengal and Orissa of India and also to exploit their potential for the production of poly(3hydroxyalkanoic acids). Altogether 15 soil samples collected from different solar salterns of coastal Orissa and West Bengal, India, were subjected to physicochemical and microbiological analyses. A total of 203 moderately halophilic bacterial cultures differing in phenotypic characteristics were isolated from solar salterns and were capable of growing optimally at 10% (w/v) NaCl in culture medium. About 54% of the isolates were found to synthesize and accumulate wide range of poly(3-hydroxybutyrate) [P(3HB)] during growth. The potent bacterial isolates were characterized in details and identified as strains of Halomonas salina, H. maura and H. marina. The cultural and nutritional conditions for efficient production of P(3HB) by the Halomonas marina HMA 103 (MTCC 8968) has been optimized in batch culture. Growth of the organism in 2% (w/v) glucose resulted in P(3HB) accumulation accounting more than 59% of cell dry weight after 50 hours incubation. The optimum P(3HB) production was attained with a combined supply of NH4 Cl and yeast extract as N-source, 0.01% (w/v) phosphate, 1.5% (w/v) sulphate and 10% (w/v) NaCl. Qualitative and quantitative 1 H NMR and FTIR analysis of cells grown in alkanoic acids (C3 —C6 ) as sole source of carbon and cosubstrates revealed synthesis of PHA copolymers composed of 3-hydroxybutyric acid and 3-hydroxyvaleric acid [P(3HB-co-3HV)]. In two-step cultivation, accumulation of the copolymer was significantly improved (80% CDW) in glucose medium supplemented with valerate (0.1%, w/v) as co-substrate S92
www.elsevier.com/locate/nbt
doi:10.1016/j.nbt.2009.06.369
2.1.123
1
Chemical Engineering Department, Middle East Technical University, Ankara, Turkey 2 Astbury Centre for Structural Molecular Biology, Institute of Molecular and Cellular Biology, University of Leeds, Leeds, United Kingdom 3 Food Engineering Department, Middle East Technical University, Ankara, Turkey
Catalases (EC 1.11.1.6) are metalloenzymes that catalyze the degradation of hydrogen peroxide (H2 O2 ) to dioxygen and water. This function is essential to control hydrogen peroxide levels in living systems, where hydrogen peroxide is a by-product of mitochondrial electron transport, -oxidation of fatty acids and photorespiration. Catalases are isolated from a broad range of prokaryotic and eukaryotic microorganisms. Fungi are effective producers of these enzymes possessing catalases, peroxidases, and superoxide dismutases to ensure that reactive oxygen species (ROS) like H2 O2 , superoxide, hydroxyl radicals, and singlet oxygen are maintained at safe concentrations. Catalases are classified into three groups: monofunctional heme (typical) catalases, catalase-peroxidases and manganese catalases. A new fourth group of catalases, the catalase-phenol oxidases (CATPO), are introduced in this study. CATPOs are bifunctional enzymes being capable of hydrogen peroxide decomposition (catalase activity) and phenolic (o-diphenolic compound, especially catechol) oxidation in the absence of hydrogen peroxide (phenol oxidase activity). We are only aware of one study describing phenol oxidase activity of a mammalian catalase; however, lack of characterization makes it difficult to classify the enzyme. In this study, a novel bifunctional catalase with an additional phenol oxidase activity was isolated from a thermophilic fungus, Scytalidium thermophilum. This extracellular enzyme was purified ca. tenfold with 46% yield by using anion exchange and gel filtration chromatography techniques and was biochemically characterized. The enzyme has a molecular weight of 320 kDa with four 80 kDa subunits and an isoelectric point of 5.0. CATPO contains heme which was determined by the peak in UV—vis spectrum at 406 nm (Soret band). The activation energies of catalase and phenol oxidase activities of the enzyme were found as 2.7 ± 0.2 and 10.1 ± 0.4 kcal/mol, respectively. CATPO shows potential for various industrial applications due to high thermal and pH stability of both catalase and phenol oxidase activities. The pure enzyme can oxidize o-diphenols such as catechol, caffeic acid, and L-DOPA in the absence of hydrogen peroxide with the highest oxidase activity against catechol. No activity of CATPO is detected against tyrosine and common laccase substrates such as ABTS and syringal-
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
ABSTRACTS
lases reported have ability to hydrolyze high concentration raw starch mashes effectively because of the inhibitory effect of the substrates or products on the enzyme activity. In the present study, we report the production, purification, characterization and partial amino acid sequences of a raw starch digesting ␣-amylase produced by Anoxybacillus amylolyticus isolated from Mount Rittmann in Antarctica.
and the polymer contained 88.1 and 12.8 mol% 3HB and 3HV monomer, respectively.
doi:10.1016/j.nbt.2009.06.368
Purification, characterization and identification of a novel bifunctional catalase-phenol oxidase from Scytalidium thermophilum
2.1.122
D. Sutay Kocabas 1,∗ , U. Bakir 1 , S.E. Phillips 2 , M.J. Mcpherson 2 , Z.B. Ogel 3
Production of poly(3-hydroxyalkanoic acids) by moderately halophilic bacteria isolated from solar salterns A.K. Paul 1,∗ , A. Biswas 2 1
2
Department of Botany, University of Calcutta, Kolkata, India University of Calcutta, Kolkata, India
Moderately halophilic bacteria capable of growing optimally at 0.5—2.5 M NaCl have long been attracted the attention of microbiologists as sources of biopolymers, more specifically the biopolyesters, poly(3-hydroxyalkanoic acid) [P(3HB)] and its copolymer, poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) [P(3HB-co-3HV)]. These polyesters represent the most extensively studied members of poly(hydroxyalkanoic acids) [PHAs] and are known to posses properties similar to petrochemical based plastics. In addition, these PHA plastics are completely biodegradable in the environment, biocompatible and could be produced from renewable sources. The present investigation is aimed at to explore the moderately halophilic heterotrophic bacteria from hypersaline environments of coastal West Bengal and Orissa of India and also to exploit their potential for the production of poly(3hydroxyalkanoic acids). Altogether 15 soil samples collected from different solar salterns of coastal Orissa and West Bengal, India, were subjected to physicochemical and microbiological analyses. A total of 203 moderately halophilic bacterial cultures differing in phenotypic characteristics were isolated from solar salterns and were capable of growing optimally at 10% (w/v) NaCl in culture medium. About 54% of the isolates were found to synthesize and accumulate wide range of poly(3-hydroxybutyrate) [P(3HB)] during growth. The potent bacterial isolates were characterized in details and identified as strains of Halomonas salina, H. maura and H. marina. The cultural and nutritional conditions for efficient production of P(3HB) by the Halomonas marina HMA 103 (MTCC 8968) has been optimized in batch culture. Growth of the organism in 2% (w/v) glucose resulted in P(3HB) accumulation accounting more than 59% of cell dry weight after 50 hours incubation. The optimum P(3HB) production was attained with a combined supply of NH4 Cl and yeast extract as N-source, 0.01% (w/v) phosphate, 1.5% (w/v) sulphate and 10% (w/v) NaCl. Qualitative and quantitative 1 H NMR and FTIR analysis of cells grown in alkanoic acids (C3 —C6 ) as sole source of carbon and cosubstrates revealed synthesis of PHA copolymers composed of 3-hydroxybutyric acid and 3-hydroxyvaleric acid [P(3HB-co-3HV)]. In two-step cultivation, accumulation of the copolymer was significantly improved (80% CDW) in glucose medium supplemented with valerate (0.1%, w/v) as co-substrate S92
www.elsevier.com/locate/nbt
doi:10.1016/j.nbt.2009.06.369
2.1.123
1
Chemical Engineering Department, Middle East Technical University, Ankara, Turkey 2 Astbury Centre for Structural Molecular Biology, Institute of Molecular and Cellular Biology, University of Leeds, Leeds, United Kingdom 3 Food Engineering Department, Middle East Technical University, Ankara, Turkey
Catalases (EC 1.11.1.6) are metalloenzymes that catalyze the degradation of hydrogen peroxide (H2 O2 ) to dioxygen and water. This function is essential to control hydrogen peroxide levels in living systems, where hydrogen peroxide is a by-product of mitochondrial electron transport, -oxidation of fatty acids and photorespiration. Catalases are isolated from a broad range of prokaryotic and eukaryotic microorganisms. Fungi are effective producers of these enzymes possessing catalases, peroxidases, and superoxide dismutases to ensure that reactive oxygen species (ROS) like H2 O2 , superoxide, hydroxyl radicals, and singlet oxygen are maintained at safe concentrations. Catalases are classified into three groups: monofunctional heme (typical) catalases, catalase-peroxidases and manganese catalases. A new fourth group of catalases, the catalase-phenol oxidases (CATPO), are introduced in this study. CATPOs are bifunctional enzymes being capable of hydrogen peroxide decomposition (catalase activity) and phenolic (o-diphenolic compound, especially catechol) oxidation in the absence of hydrogen peroxide (phenol oxidase activity). We are only aware of one study describing phenol oxidase activity of a mammalian catalase; however, lack of characterization makes it difficult to classify the enzyme. In this study, a novel bifunctional catalase with an additional phenol oxidase activity was isolated from a thermophilic fungus, Scytalidium thermophilum. This extracellular enzyme was purified ca. tenfold with 46% yield by using anion exchange and gel filtration chromatography techniques and was biochemically characterized. The enzyme has a molecular weight of 320 kDa with four 80 kDa subunits and an isoelectric point of 5.0. CATPO contains heme which was determined by the peak in UV—vis spectrum at 406 nm (Soret band). The activation energies of catalase and phenol oxidase activities of the enzyme were found as 2.7 ± 0.2 and 10.1 ± 0.4 kcal/mol, respectively. CATPO shows potential for various industrial applications due to high thermal and pH stability of both catalase and phenol oxidase activities. The pure enzyme can oxidize o-diphenols such as catechol, caffeic acid, and L-DOPA in the absence of hydrogen peroxide with the highest oxidase activity against catechol. No activity of CATPO is detected against tyrosine and common laccase substrates such as ABTS and syringal-
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