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Wolfgang Voelter - One of the best experts on this subject based on the ideXlab platform.
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spectroscopic properties of a novel Neutral Proteinase from saccharomonospora canescens
Comparative Biochemistry and Physiology B, 2000Co-Authors: Pavlina Dolashkaangelova, Stanka Stoeva, Wolfgang VoelterAbstract:Abstract A Neutral Proteinase (NPS) was purified from the culture broth of Saccharomonospora canescens sp. novus , strain 5, using DEAE cellulose and a POROS HQ/M 4.6×100 mm column. The stability towards thermal and chemical (guanidine hydrochloride, Gdn.HCl) denaturation of NPS was investigated by kinetic and equilibrium studies. The unfolding processes were monitored by circular dichroism and fluorescence spectroscopy. The free energy of stabilization in water was calculated to be 2.1 kcal mol −1 . The thermostability was determined by the critical temperature T c from fluorescence measurements (69°C) and the melting temperature T m (70°C) from (1) measurements. Quenching with acrylamide, iodide and cesium gives information about the microenvironment of intrinsic protein fluorophores. The K sv constant for NPS is 4.6 and classifies the emitting tryptophans as ‘buried’ in the hydrophobic interior of the investigated protein.
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A novel thermostable Neutral Proteinase from Saccharomonospora canescens
Biochimica et biophysica acta, 1998Co-Authors: Pavlina Dolashka, Dessislava Georgieva, Stanka Stoeva, Nicolay Genov, Rossen Rachev, Adriana Gusterova, Wolfgang VoelterAbstract:Abstract A novel thermostable Neutral Proteinase, called NPS, was purified to electrophoretic homogeneity from the culture broth of Saccharomonospora canescens sp. novus, strain 5. The molecular mass was determined by SDS-polyacrylamide gel electrophoresis to be 35 000 Da. The enzyme exhibits a sharp pH optimum of proteolytic activity at pH 6.7. NPS was completely inactivated with inhibitors, typical for metalloendopeptidases, EDTA and 1,10-phenantroline, whereas the serine Proteinase inhibitor PMSF had no effect. Atomic absorption measurements showed that the Proteinase binds a single zinc and four calcium ions. The enzyme thermostability was characterized in the absence and presence of added calcium. Melting temperature, T m =77°C and an activation energy, E a , for the thermal deactivation of the excited protein fluorophores of 72.13 kJ mol −1 were calculated in the presence of 100 mM CaCl 2 . The E a -value is considerably higher than those obtained for a number of Proteinases from microorganisms and was explained by the thermostable structure of the enzyme. Effective radiationless energy transfer from phenol groups to indole rings was observed. 68% of the light absorbed by tyrosyl residues is transfered to tryptophyl side chains. No homology was found after comparison of the NPS N-terminal sequence, including the first 26 residues, with those of other Neutral Proteinases from microorganisms. In contrast to the well-known bacterial Neutral Proteinase thermolysin and related enzymes from microorganisms, NPS possesses arylamidase and esterase activities. Further crystallographic studies will reveal the structural reasons for this specificity. Epoxy and epithio pyranosides are inhibitors of the Proteinase arylamidase activity.
Peter L Bergquist - One of the best experts on this subject based on the ideXlab platform.
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sequence of the gene encoding a highly thermostable Neutral Proteinase from bacillus sp strain ea1 expression in escherichia coli and characterisation
Biochimica et Biophysica Acta, 1996Co-Authors: David J Saul, Liam Williams, Helen S Toogood, Roy M Daniel, Peter L BergquistAbstract:The gene for a highly thermostable Neutral Proteinase (Npr) was isolated from Bacillus sp. strain EA1 by the polymerase chain reaction using consensus primers based on the sequences of npr genes from related species. The gene was sequenced and shown to be closely related to a Neutral Proteinase gene from Bacillus caldolyticus strain YP-T; the mature form of the enzyme differing by only a single amino acid. Enzyme samples were prepared from both the native organisms and also from recombinant Escherichia coli expressing the two npr genes. The Proteinase from strain EA1 was shown to be significantly more thermostable than that from B. caldolyticus and that this difference is the result of a single amino acid substitution which is situated proximal to a region of the enzyme known to be crucial to conferring thermal stability. The phylogenetic relationship of EA1 to other Bacilli is also described.
Pavlina Dolashka - One of the best experts on this subject based on the ideXlab platform.
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A novel thermostable Neutral Proteinase from Saccharomonospora canescens
Biochimica et biophysica acta, 1998Co-Authors: Pavlina Dolashka, Dessislava Georgieva, Stanka Stoeva, Nicolay Genov, Rossen Rachev, Adriana Gusterova, Wolfgang VoelterAbstract:Abstract A novel thermostable Neutral Proteinase, called NPS, was purified to electrophoretic homogeneity from the culture broth of Saccharomonospora canescens sp. novus, strain 5. The molecular mass was determined by SDS-polyacrylamide gel electrophoresis to be 35 000 Da. The enzyme exhibits a sharp pH optimum of proteolytic activity at pH 6.7. NPS was completely inactivated with inhibitors, typical for metalloendopeptidases, EDTA and 1,10-phenantroline, whereas the serine Proteinase inhibitor PMSF had no effect. Atomic absorption measurements showed that the Proteinase binds a single zinc and four calcium ions. The enzyme thermostability was characterized in the absence and presence of added calcium. Melting temperature, T m =77°C and an activation energy, E a , for the thermal deactivation of the excited protein fluorophores of 72.13 kJ mol −1 were calculated in the presence of 100 mM CaCl 2 . The E a -value is considerably higher than those obtained for a number of Proteinases from microorganisms and was explained by the thermostable structure of the enzyme. Effective radiationless energy transfer from phenol groups to indole rings was observed. 68% of the light absorbed by tyrosyl residues is transfered to tryptophyl side chains. No homology was found after comparison of the NPS N-terminal sequence, including the first 26 residues, with those of other Neutral Proteinases from microorganisms. In contrast to the well-known bacterial Neutral Proteinase thermolysin and related enzymes from microorganisms, NPS possesses arylamidase and esterase activities. Further crystallographic studies will reveal the structural reasons for this specificity. Epoxy and epithio pyranosides are inhibitors of the Proteinase arylamidase activity.
Stanka Stoeva - One of the best experts on this subject based on the ideXlab platform.
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spectroscopic properties of a novel Neutral Proteinase from saccharomonospora canescens
Comparative Biochemistry and Physiology B, 2000Co-Authors: Pavlina Dolashkaangelova, Stanka Stoeva, Wolfgang VoelterAbstract:Abstract A Neutral Proteinase (NPS) was purified from the culture broth of Saccharomonospora canescens sp. novus , strain 5, using DEAE cellulose and a POROS HQ/M 4.6×100 mm column. The stability towards thermal and chemical (guanidine hydrochloride, Gdn.HCl) denaturation of NPS was investigated by kinetic and equilibrium studies. The unfolding processes were monitored by circular dichroism and fluorescence spectroscopy. The free energy of stabilization in water was calculated to be 2.1 kcal mol −1 . The thermostability was determined by the critical temperature T c from fluorescence measurements (69°C) and the melting temperature T m (70°C) from (1) measurements. Quenching with acrylamide, iodide and cesium gives information about the microenvironment of intrinsic protein fluorophores. The K sv constant for NPS is 4.6 and classifies the emitting tryptophans as ‘buried’ in the hydrophobic interior of the investigated protein.
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A novel thermostable Neutral Proteinase from Saccharomonospora canescens
Biochimica et biophysica acta, 1998Co-Authors: Pavlina Dolashka, Dessislava Georgieva, Stanka Stoeva, Nicolay Genov, Rossen Rachev, Adriana Gusterova, Wolfgang VoelterAbstract:Abstract A novel thermostable Neutral Proteinase, called NPS, was purified to electrophoretic homogeneity from the culture broth of Saccharomonospora canescens sp. novus, strain 5. The molecular mass was determined by SDS-polyacrylamide gel electrophoresis to be 35 000 Da. The enzyme exhibits a sharp pH optimum of proteolytic activity at pH 6.7. NPS was completely inactivated with inhibitors, typical for metalloendopeptidases, EDTA and 1,10-phenantroline, whereas the serine Proteinase inhibitor PMSF had no effect. Atomic absorption measurements showed that the Proteinase binds a single zinc and four calcium ions. The enzyme thermostability was characterized in the absence and presence of added calcium. Melting temperature, T m =77°C and an activation energy, E a , for the thermal deactivation of the excited protein fluorophores of 72.13 kJ mol −1 were calculated in the presence of 100 mM CaCl 2 . The E a -value is considerably higher than those obtained for a number of Proteinases from microorganisms and was explained by the thermostable structure of the enzyme. Effective radiationless energy transfer from phenol groups to indole rings was observed. 68% of the light absorbed by tyrosyl residues is transfered to tryptophyl side chains. No homology was found after comparison of the NPS N-terminal sequence, including the first 26 residues, with those of other Neutral Proteinases from microorganisms. In contrast to the well-known bacterial Neutral Proteinase thermolysin and related enzymes from microorganisms, NPS possesses arylamidase and esterase activities. Further crystallographic studies will reveal the structural reasons for this specificity. Epoxy and epithio pyranosides are inhibitors of the Proteinase arylamidase activity.
Adriana Gusterova - One of the best experts on this subject based on the ideXlab platform.
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A novel thermostable Neutral Proteinase from Saccharomonospora canescens
Biochimica et biophysica acta, 1998Co-Authors: Pavlina Dolashka, Dessislava Georgieva, Stanka Stoeva, Nicolay Genov, Rossen Rachev, Adriana Gusterova, Wolfgang VoelterAbstract:Abstract A novel thermostable Neutral Proteinase, called NPS, was purified to electrophoretic homogeneity from the culture broth of Saccharomonospora canescens sp. novus, strain 5. The molecular mass was determined by SDS-polyacrylamide gel electrophoresis to be 35 000 Da. The enzyme exhibits a sharp pH optimum of proteolytic activity at pH 6.7. NPS was completely inactivated with inhibitors, typical for metalloendopeptidases, EDTA and 1,10-phenantroline, whereas the serine Proteinase inhibitor PMSF had no effect. Atomic absorption measurements showed that the Proteinase binds a single zinc and four calcium ions. The enzyme thermostability was characterized in the absence and presence of added calcium. Melting temperature, T m =77°C and an activation energy, E a , for the thermal deactivation of the excited protein fluorophores of 72.13 kJ mol −1 were calculated in the presence of 100 mM CaCl 2 . The E a -value is considerably higher than those obtained for a number of Proteinases from microorganisms and was explained by the thermostable structure of the enzyme. Effective radiationless energy transfer from phenol groups to indole rings was observed. 68% of the light absorbed by tyrosyl residues is transfered to tryptophyl side chains. No homology was found after comparison of the NPS N-terminal sequence, including the first 26 residues, with those of other Neutral Proteinases from microorganisms. In contrast to the well-known bacterial Neutral Proteinase thermolysin and related enzymes from microorganisms, NPS possesses arylamidase and esterase activities. Further crystallographic studies will reveal the structural reasons for this specificity. Epoxy and epithio pyranosides are inhibitors of the Proteinase arylamidase activity.