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KMID : 1007519970060010008
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Food Science and Biotechnology
1997 Volume.6 No. 1 p.8 ~ p.11
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Purification and Characterization of D - Xylose Isomerase in Recombinant Escherichia coli
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Rhee, In Koo
Joo, Gil Jae/Park, Heui Dong
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Abstract
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The D-xylose isomerase gene (xylA) cloned previously from Escherichia coli K802 was overexpressed by using the λPL promoter in E. coli M5248 which carried the λcI857 gene on the chromosomal DNA. The transformants (E. coli M5248/pPX152) produecd 15 times as much D-xylose isomerase as that of parent strain E. coli K802, and the amount of overproduced Dxylose isomerase was found to be about 60% of total protein in cell-free extracts (11). The overproduced D-xylose isomerase was purified to homogeneity from cell-lysate by chromatography with DEAE-cellulose, DEAE-sephacel, DEAE-sephadex A-50, and Sephadex G-200 column. The molecular weight of the purified enzyme was estimated to be 44 kDa by SDS-PAGE and 88 kDa by gel filtration, indicating that this enzyme behaved as a dimer. The optimal pH and temperature were 7.5 and 37℃, respectively. Also we were observed the inclusion bodies in cells by using electron microscope. The solubilized enzymes by 8M urea from inclusion bodies were found to have no D-xylose isomerase activity, but had crossreacting activity with antiserum against the purified D-xylose isomerase in double immunodiffusion assay.
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