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  • Purification, Peptide Sequencing, Modelling and Characterisation of Extracellular Laccase Secreted by Pleurotus Sajorcaju MTCC 141

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    Dr. Antik Bose
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    The extracellular laccase which is secreted by Pleurotus sajorcaju has been purified using Ammonium sulphate precipitation, DEAE-cellulose, Sephadex G-100, ConA-Sepharose 4B and HP1090A HPLC fitted with Vydac C-18 Reverse- Phase chromatography with 722.2 fold of purification and yield of 1.64%. 12% SDS-PAGE showed a monomer of 90KD. Western Blotting was done with monoclonal Lcc Cbr2 rabbit antibody raised against copper binding region II of laccase from Agaricus bisporus. Peptide sequencing of the purified laccase showed 529 amino acids long peptide with a 23 amino acids long signal peptide. Three Plastocyanin like domains were observed ( 25-159, 170-312 and 380-499). Residues 57, 239, 282 and 465 of the extracellular laccase showed N- linked N-acetyl D-glucoseamine residues. The enzyme showed KM of 36µM, 216pM, 380pM, 370pM and 260pM with 2,6 dimethoxyphenol, Catechol, m-cresol, pyrogallol and syringaldazine respectively. CuSo4, BaCl2, MgCl2, FeCL3, ZnCl3, Urea, PCMB, DTT, β-mercaptoethanol had no effect on enzyme activity while HgCl2 and MnCl2 were weak inhibitors and SDS, Sodium azide were strong inhibitors. Crystallization data, collection, synchrotron radiation andThe extracellular laccase which is secreted by Pleurotus sajorcaju has been purified using Ammonium sulphate precipitation, DEAE-cellulose, Sephadex G-100, ConA-Sepharose 4B and HP1090A HPLC fitted with Vydac C-18 Reverse- Phase chromatography with 722.2 fold of purification and yield of 1.64%. 12% SDS-PAGE showed a monomer of 90KD. Western Blotting was done with monoclonal Lcc Cbr2 rabbit antibody raised against copper binding region II of laccase from Agaricus bisporus. Peptide sequencing of the purified laccase showed 529 amino acids long peptide with a 23 amino acids long signal peptide. Three Plastocyanin like domains were observed ( 25-159, 170-312 and 380-499). Residues 57, 239, 282 and 465 of the extracellular laccase showed N- linked N-acetyl D-glucoseamine residues. The enzyme showed KM of 36µM, 216pM, 380pM, 370pM and 260pM with 2,6 dimethoxyphenol, Catechol, m-cresol, pyrogallol and syringaldazine respectively. CuSo4, BaCl2, MgCl2, FeCL3, ZnCl3, Urea, PCMB, DTT, β-mercaptoethanol had no effect on enzyme activity while HgCl2 and MnCl2 were weak inhibitors and SDS, Sodium azide were strong inhibitors. Crystallization data, collection, synchrotron radiation andThe extracellular laccase which is secreted by Pleurotus sajorcaju has been purified using Ammonium sulphate precipitation, DEAE-cellulose, Sephadex G-100, ConA-Sepharose 4B and HP1090A HPLC fitted with Vydac C-18 Reverse- Phase chromatography with 722.2 fold of purification and yield of 1.64%. 12% SDS-PAGE showed a monomer of 90KD. Western Blotting was done with monoclonal Lcc Cbr2 rabbit antibody raised against copper binding region II of laccase from Agaricus bisporus. Peptide sequencing of the purified laccase showed 529 amino acids long peptide with a 23 amino acids long signal peptide. Three Plastocyanin like domains were observed ( 25-159, 170-312 and 380-499). Residues 57, 239, 282 and 465 of the extracellular laccase showed N- linked N-acetyl D-glucoseamine residues. The enzyme showed KM of 36µM, 216pM, 380pM, 370pM and 260pM with 2,6 dimethoxyphenol, Catechol, m-cresol, pyrogallol and syringaldazine respectively. CuSo4, BaCl2, MgCl2, FeCL3, ZnCl3, Urea, PCMB, DTT, β-mercaptoethanol had no effect on enzyme activity while HgCl2 and MnCl2 were weak inhibitors and SDS, Sodium azide were strong inhibitors. Crystallization data, collection, synchrotron radiation and processing have been carried out. Orthorhombic crystals of space group P2 12 121 were obtained with dimensions a= 83.6 ͦA, b= 85.0 ͦA, c= 91.5 ͦA with corresponding Vm of 2.3 ͦA 3/Da, assuming one molecule per asymmetric unit. Diffraction data was collected using Beamline BW7B of Synchroton Deutoches Elektonen Synchroton (DESY) at room temperature. Data was processed and scaled with programs DENZO and SCALEPACK of Hkl suit. For

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    The extracellular laccase which is secreted by Pleurotus sajorcaju has been purified using Ammonium sulphate precipitation, DEAE-cellulose, Sephadex G-100, ConA-Sepharose 4B and HP1090A HPLC fitted with Vydac C-18 Reverse- Phase chromatography with 722.2 fold of purification and yield of 1.64%. 12% SDS-PAGE showed a monomer of 90KD. Western Blotting was done with monoclonal Lcc Cbr2 rabbit antibody raised against copper binding region II of laccase from Agaricus bisporus. Peptide sequencing of the purified laccase showed 529 amino acids long peptide with a 23 amino acids long signal peptide. Three Plastocyanin like domains were observed ( 25-159, 170-312 and 380-499). Residues 57, 239, 282 and 465 of the extracellular laccase showed N- linked N-acetyl D-glucoseamine residues. The enzyme showed KM of 36µM, 216pM, 380pM, 370pM and 260pM with 2,6 dimethoxyphenol, Catechol, m-cresol, pyrogallol and syringaldazine respectively. CuSo4, BaCl2, MgCl2, FeCL3, ZnCl3, Urea, PCMB, DTT, β-mercaptoethanol had no effect on enzyme activity while HgCl2 and MnCl2 were weak inhibitors and SDS, Sodium azide were strong inhibitors. Crystallization data, collection, synchrotron radiation andThe extracellular laccase which is secreted by Pleurotus sajorcaju has been purified using Ammonium sulphate precipitation, DEAE-cellulose, Sephadex G-100, ConA-Sepharose 4B and HP1090A HPLC fitted with Vydac C-18 Reverse- Phase chromatography with 722.2 fold of purification and yield of 1.64%. 12% SDS-PAGE showed a monomer of 90KD. Western Blotting was done with monoclonal Lcc Cbr2 rabbit antibody raised against copper binding region II of laccase from Agaricus bisporus. Peptide sequencing of the purified laccase showed 529 amino acids long peptide with a 23 amino acids long signal peptide. Three Plastocyanin like domains were observed ( 25-159, 170-312 and 380-499). Residues 57, 239, 282 and 465 of the extracellular laccase showed N- linked N-acetyl D-glucoseamine residues. The enzyme showed KM of 36µM, 216pM, 380pM, 370pM and 260pM with 2,6 dimethoxyphenol, Catechol, m-cresol, pyrogallol and syringaldazine respectively. CuSo4, BaCl2, MgCl2, FeCL3, ZnCl3, Urea, PCMB, DTT, β-mercaptoethanol had no effect on enzyme activity while HgCl2 and MnCl2 were weak inhibitors and SDS, Sodium azide were strong inhibitors. Crystallization data, collection, synchrotron radiation andThe extracellular laccase which is secreted by Pleurotus sajorcaju has been purified using Ammonium sulphate precipitation, DEAE-cellulose, Sephadex G-100, ConA-Sepharose 4B and HP1090A HPLC fitted with Vydac C-18 Reverse- Phase chromatography with 722.2 fold of purification and yield of 1.64%. 12% SDS-PAGE showed a monomer of 90KD. Western Blotting was done with monoclonal Lcc Cbr2 rabbit antibody raised against copper binding region II of laccase from Agaricus bisporus. Peptide sequencing of the purified laccase showed 529 amino acids long peptide with a 23 amino acids long signal peptide. Three Plastocyanin like domains were observed ( 25-159, 170-312 and 380-499). Residues 57, 239, 282 and 465 of the extracellular laccase showed N- linked N-acetyl D-glucoseamine residues. The enzyme showed KM of 36µM, 216pM, 380pM, 370pM and 260pM with 2,6 dimethoxyphenol, Catechol, m-cresol, pyrogallol and syringaldazine respectively. CuSo4, BaCl2, MgCl2, FeCL3, ZnCl3, Urea, PCMB, DTT, β-mercaptoethanol had no effect on enzyme activity while HgCl2 and MnCl2 were weak inhibitors and SDS, Sodium azide were strong inhibitors. Crystallization data, collection, synchrotron radiation and processing have been carried out. Orthorhombic crystals of space group P2 12 121 were obtained with dimensions a= 83.6 ͦA, b= 85.0 ͦA, c= 91.5 ͦA with corresponding Vm of 2.3 ͦA 3/Da, assuming one molecule per asymmetric unit. Diffraction data was collected using Beamline BW7B of Synchroton Deutoches Elektonen Synchroton (DESY) at room temperature. Data was processed and scaled with programs DENZO and SCALEPACK of Hkl suit. For

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    40 views2 pages

    Purification, Peptide Sequencing, Modelling and Characterisation of Extracellular Laccase Secreted by Pleurotus Sajorcaju MTCC 141

    Uploaded by

    Dr. Antik Bose
    The extracellular laccase which is secreted by Pleurotus sajorcaju has been purified using Ammonium sulphate precipitation, DEAE-cellulose, Sephadex G-100, ConA-Sepharose 4B and HP1090A HPLC fitted with Vydac C-18 Reverse- Phase chromatography with 722.2 fold of purification and yield of 1.64%. 12% SDS-PAGE showed a monomer of 90KD. Western Blotting was done with monoclonal Lcc Cbr2 rabbit antibody raised against copper binding region II of laccase from Agaricus bisporus. Peptide sequencing of the purified laccase showed 529 amino acids long peptide with a 23 amino acids long signal peptide. Three Plastocyanin like domains were observed ( 25-159, 170-312 and 380-499). Residues 57, 239, 282 and 465 of the extracellular laccase showed N- linked N-acetyl D-glucoseamine residues. The enzyme showed KM of 36µM, 216pM, 380pM, 370pM and 260pM with 2,6 dimethoxyphenol, Catechol, m-cresol, pyrogallol and syringaldazine respectively. CuSo4, BaCl2, MgCl2, FeCL3, ZnCl3, Urea, PCMB, DTT, β-mercaptoethanol had no effect on enzyme activity while HgCl2 and MnCl2 were weak inhibitors and SDS, Sodium azide were strong inhibitors. Crystallization data, collection, synchrotron radiation andThe extracellular laccase which is secreted by Pleurotus sajorcaju has been purified using Ammonium sulphate precipitation, DEAE-cellulose, Sephadex G-100, ConA-Sepharose 4B and HP1090A HPLC fitted with Vydac C-18 Reverse- Phase chromatography with 722.2 fold of purification and yield of 1.64%. 12% SDS-PAGE showed a monomer of 90KD. Western Blotting was done with monoclonal Lcc Cbr2 rabbit antibody raised against copper binding region II of laccase from Agaricus bisporus. Peptide sequencing of the purified laccase showed 529 amino acids long peptide with a 23 amino acids long signal peptide. Three Plastocyanin like domains were observed ( 25-159, 170-312 and 380-499). Residues 57, 239, 282 and 465 of the extracellular laccase showed N- linked N-acetyl D-glucoseamine residues. The enzyme showed KM of 36µM, 216pM, 380pM, 370pM and 260pM with 2,6 dimethoxyphenol, Catechol, m-cresol, pyrogallol and syringaldazine respectively. CuSo4, BaCl2, MgCl2, FeCL3, ZnCl3, Urea, PCMB, DTT, β-mercaptoethanol had no effect on enzyme activity while HgCl2 and MnCl2 were weak inhibitors and SDS, Sodium azide were strong inhibitors. Crystallization data, collection, synchrotron radiation andThe extracellular laccase which is secreted by Pleurotus sajorcaju has been purified using Ammonium sulphate precipitation, DEAE-cellulose, Sephadex G-100, ConA-Sepharose 4B and HP1090A HPLC fitted with Vydac C-18 Reverse- Phase chromatography with 722.2 fold of purification and yield of 1.64%. 12% SDS-PAGE showed a monomer of 90KD. Western Blotting was done with monoclonal Lcc Cbr2 rabbit antibody raised against copper binding region II of laccase from Agaricus bisporus. Peptide sequencing of the purified laccase showed 529 amino acids long peptide with a 23 amino acids long signal peptide. Three Plastocyanin like domains were observed ( 25-159, 170-312 and 380-499). Residues 57, 239, 282 and 465 of the extracellular laccase showed N- linked N-acetyl D-glucoseamine residues. The enzyme showed KM of 36µM, 216pM, 380pM, 370pM and 260pM with 2,6 dimethoxyphenol, Catechol, m-cresol, pyrogallol and syringaldazine respectively. CuSo4, BaCl2, MgCl2, FeCL3, ZnCl3, Urea, PCMB, DTT, β-mercaptoethanol had no effect on enzyme activity while HgCl2 and MnCl2 were weak inhibitors and SDS, Sodium azide were strong inhibitors. Crystallization data, collection, synchrotron radiation and processing have been carried out. Orthorhombic crystals of space group P2 12 121 were obtained with dimensions a= 83.6 ͦA, b= 85.0 ͦA, c= 91.5 ͦA with corresponding Vm of 2.3 ͦA 3/Da, assuming one molecule per asymmetric unit. Diffraction data was collected using Beamline BW7B of Synchroton Deutoches Elektonen Synchroton (DESY) at room temperature. Data was processed and scaled with programs DENZO and SCALEPACK of Hkl suit. For

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    Attribution Non-Commercial (BY-NC)
    Download as PDF, TXT or read online from Scribd
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    Purification, peptide sequencing, modelling and characterization of extracellular laccase secreted by Pleurotus sajor-caju MTCC 141

    Antik Bose

    Affilations I Contributions I Corresponding authors

    The extracellular laccase which is secreted by Pleurotus sajorcaju has been purified using Ammonium sulphate precipitation, DEAE-cellulose, Sephadex G-100, ConA-Sepharose 4B and HP1090A HPLC fitted with Vydac C-18 Reverse- Phase chromatography with 722.2 fold of purification and yield of 1.64%. 12% SDS-PAGE showed a monomer of 90KD. Western Blotting was done with monoclonal Lcc Cbr2 rabbit antibody raised against copper binding region II of laccase from Agaricus bisporus. Peptide sequencing of the purified laccase showed 529 amino acids long peptide with a 23 amino acids long signal peptide. Three Plastocyanin like domains were observed ( 25-159, 170-312 and 380-499). Residues 57, 239, 282 and 465 of the extracellular laccase showed Nlinked N-acetyl D-glucoseamine residues. The enzyme showed KM of 36M, 216pM, 380pM, 370pM and 260pM with 2,6 dimethoxyphenol, Catechol, m-cresol, pyrogallol and syringaldazine respectively. CuSo4, BaCl2, MgCl2, FeCL3, ZnCl3, Urea, PCMB, DTT, -mercaptoethanol had no effect on enzyme activity while HgCl2 and MnCl2 were weak inhibitors and SDS, Sodium azide were strong inhibitors. Crystallization data, collection, synchrotron radiation and processing have been carried out. Orthorhombic crystals of space group P2 12 121 w A, b= 85.0 A, c= 91.5 A 3/Da, assuming one molecule per asymmetric unit. Diffraction data was collected using Beamline BW7B of Synchroton Deutoches Elektonen Synchroton (DESY) at room temperature. Data was processed and scaled with programs DENZO and SCALEPACK of Hkl suit. For 1.9-20 A resolution shell, the data were 99.7% complete and scale with an overall Rsym of 0.063 and Rsym of 0.38 for data in highest resolution A resolution data, an Rcryst of 0.434 was obtained. A 2F 0Fe electron density map revealed clearly all four copper sites . Repeated rounds of refinement with REFMAC resulted in a complete and well-defined model with an Rwork A resolution. Only 4 out of total 499 amino acids were in generously allowed region of Ramachandran Plot.

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