Studies on the modification and enzymatic hydrolysis of poly (y-D-glutamic acid) from Bacillus licheniformis 9945A
Studies on the modification and enzymatic hydrolysis of poly (y-D-glutamic acid) from Bacillus licheniformis 9945A
Poly (γ-D-glutamic acid) is a bacterial capsular polymer produced by Bacillus licheniformis (Troy, F.A. J. Biol. Chem. 1973, 248, 305 - 315). The polymer has an unusual structure in that it is composed of the gamma-linked D-isomer of glutamic acid monomers (Troy, F.A. J. Biol. Chem. 1973 248, 316 - 324). Poly (γ-D-glutamic acid) has been isolated from Bacillus licheniformis 9945a and characterised by NMR spectroscopy. The molecular mass of the polymer was investigated by SDS-PAGE, gel filtration chromatography and MALDI-TOF mass spectroscopy.
Covalent modifications of poly (γ-D-glutamic acid) in aqueous solution have been studied. Activation of the alpha-carboxylate using a water soluble carbodiimide and derivatisation utilising a synthetic UV absorbent amine and ethanolamine produced a water-soluble modified polymer. Characterisation of the modified polymer indicated that cleavage of the polymeric backbone, with a reduction of molecular mass from 129 kDa to 10 kDa, occurred concomitantly with derivatisation. A procedure was developed for the removal of non-covalently bound ligands by treatment with 5 M calcium chloride. Reaction of the side chains with chalcone and 4-nitrophenylacetate to form covalent linkages was investigated.
The presence of an enzyme responsible for the degradation of poly (γ-D-glutamic acid) was located in the extracellular medium, forming a strong non-covalent interaction with poly (γ-D-glutamic acid). The enzyme can be removed from the polymer by treatment with the anionic detergent sodium dodecyl sulphate and the solubilised enzyme separated from the polymer by affinity chromatography. The enzyme has a requirement of divalent metal ions and shows maximum activity with zinc (II) ions. Activity is abolished by treatment with the metal chelator EDTA. Modification of specific amino acid residues indicates the importance of lysine, glutamic acid and histidine residues for catalytic activity. Analysis of the degradation products of the enzyme suggest that degradation of the polymer occurs by endo-cleavage. Depolymerase activity is favoured by high pH. From this data, poly (γ-D-glutamyl) depolymerase is tentatively assigned as a metallo-endo depolymerase.
University of Southampton
1998
King, Elizabeth Caroline
(1998)
Studies on the modification and enzymatic hydrolysis of poly (y-D-glutamic acid) from Bacillus licheniformis 9945A.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
Poly (γ-D-glutamic acid) is a bacterial capsular polymer produced by Bacillus licheniformis (Troy, F.A. J. Biol. Chem. 1973, 248, 305 - 315). The polymer has an unusual structure in that it is composed of the gamma-linked D-isomer of glutamic acid monomers (Troy, F.A. J. Biol. Chem. 1973 248, 316 - 324). Poly (γ-D-glutamic acid) has been isolated from Bacillus licheniformis 9945a and characterised by NMR spectroscopy. The molecular mass of the polymer was investigated by SDS-PAGE, gel filtration chromatography and MALDI-TOF mass spectroscopy.
Covalent modifications of poly (γ-D-glutamic acid) in aqueous solution have been studied. Activation of the alpha-carboxylate using a water soluble carbodiimide and derivatisation utilising a synthetic UV absorbent amine and ethanolamine produced a water-soluble modified polymer. Characterisation of the modified polymer indicated that cleavage of the polymeric backbone, with a reduction of molecular mass from 129 kDa to 10 kDa, occurred concomitantly with derivatisation. A procedure was developed for the removal of non-covalently bound ligands by treatment with 5 M calcium chloride. Reaction of the side chains with chalcone and 4-nitrophenylacetate to form covalent linkages was investigated.
The presence of an enzyme responsible for the degradation of poly (γ-D-glutamic acid) was located in the extracellular medium, forming a strong non-covalent interaction with poly (γ-D-glutamic acid). The enzyme can be removed from the polymer by treatment with the anionic detergent sodium dodecyl sulphate and the solubilised enzyme separated from the polymer by affinity chromatography. The enzyme has a requirement of divalent metal ions and shows maximum activity with zinc (II) ions. Activity is abolished by treatment with the metal chelator EDTA. Modification of specific amino acid residues indicates the importance of lysine, glutamic acid and histidine residues for catalytic activity. Analysis of the degradation products of the enzyme suggest that degradation of the polymer occurs by endo-cleavage. Depolymerase activity is favoured by high pH. From this data, poly (γ-D-glutamyl) depolymerase is tentatively assigned as a metallo-endo depolymerase.
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Published date: 1998
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Local EPrints ID: 463538
URI: http://eprints.soton.ac.uk/id/eprint/463538
PURE UUID: 8d9c4726-1687-42d0-b84e-062da6f4ec6e
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Date deposited: 04 Jul 2022 20:53
Last modified: 04 Jul 2022 20:53
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Author:
Elizabeth Caroline King
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