A mutant phospholipase D with enhanced thermostability from Streptomyces sp. |
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Authors: | Tadashi Hatanaka Tomofumi Negishi Koichi Mori |
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Institution: | 1. Research Institute for Biological Sciences, Okayama (RIBS), 7549-1 Kayo-cho, Jyobo-gun, Okayama, 716-1241, Japan;2. Research Institute of Innovative Technology for the Earth (RITE), 9-2 Kizugawadai, Kizu-cho, Soraku-gun, Kyoto, 619-0292, Japan |
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Abstract: | To investigate the contribution of amino acid residues to the thermostability of phospholipase D (PLD), a chimeric form of two Streptomyces PLDs (thermolabile K1PLD and thermostable TH-2PLD) was constructed. K/T/KPLD, in which residues 329–441 of K1PLD were recombined with the homologous region of TH-2PLD, showed a thermostability midway between those of K1PLD and TH-2PLD. By comparing the primary structures of Streptomyces PLDs, the seven candidates of thermostability-related amino acid residues of K1PLD were identified. The K1E346DPLD mutant, in which Glu346 of K1PLD was substituted with Asp by site-directed mutagenesis, exhibited enhanced thermostability, which was almost the same as that of TH-2PLD. |
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Keywords: | Phospholipase D Thermostability Biocatalyst PLD phospholipase D PpNP SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis CD circular dichroism |
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