Co-refolding of two peptide fragments derived from Agrobacterium tumefaciens beta-glucosidase with catalytic activity |
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Authors: | Kim Bong-Jo Mangala Selanere L Hayashi Kiyoshi |
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Institution: | Enzyme Laboratory, National Food Research Institute, 2-1-12 Kannondai, Tsukuba 305-8642, Japan |
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Abstract: | Four sites of the non-homologous region (coding amino acid residues of 347, 421, 466 and 533) of a gene were randomly selected for splitting to investigate the function of β-glucosidase from Agrobacterium tumefaciens in the co-refolding of peptides into the catalytically active enzyme. As a result of gene splitting, four N- and C-terminal domain peptides were obtained as insoluble inclusion bodies. No catalytic activity was observed when these fragments refolded individually. However, a considerable amount of activity was restored when the two fragments derived from N- and C- terminal peptides were co-refolded together. The deletion of amino acid residues in the non-homologous region resulted in a complete loss of enzyme activity, which suggests that truncation of amino acids in this region strongly affects the co-refolding ability of the enzyme to maintain activity. |
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Keywords: | MOPS 3-[N-morpholino] propanesulfonic acid pNP p-nitrophenyl SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis |
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