Purification and cloning of a thermostable xylose (glucose) isomerase with an acidic pH optimum from Thermoanaerobacterium strain JW/SL-YS 489
- Univ. of Georgia, Athens, GA (United States)
An unusual xylose isomerase produced by Thermoanaerobacterium strain JW/SL-YS 489 was purified 28-fold to gel electrophoretic homogeneity, and the biochemical properties were determined. Its pH optimum distinguishes this enzyme from all other previously described xylose isomerases. The purified enzyme had maximal activity at pH 6.4 (60{degrees}C) or pH 6.8 (80{degrees}) in a 30-min assay, an isoelectric point at 4.7, and an estimated native molecular mass of 200 kDa, with four identical subunits of 50 kDa. Like other xylose isomerases, this enzyme required Mn{sup 2+}, Co{sup 2+}, or Mg{sup 2+} for thermal stability (stable for 1 h at 82{degrees}C in the absence of substrate) and isomerase activity, and it preferred xylose as a substrate. the gene encoding in xylose isomerase was cloned and expressed in Escherichia coli, and the complete nucleotide sequence was determined. Analysis of the sequence revealed an open reading frame of 1,317 bp that encoded a protein of 439 amino acid residues with a calculated molecular mass of 50 kDa. The biochemical properties of the cloned enzyme were the same as those of the native enzyme. Comparison of the deduced amino acid sequence with sequences of other xylose related bacterium, Thermoanaerobacterium saccharolyticum B6A-RI. In fact, only seven amino acid differences were detected between the two sequences, and the biochemical properties of the two enzymes, except for the pH optimum, are quite similar. Both enzymes had a temperature optimum at 80{degrees}C, very similar isoelectric points (pH for strain JW/SL-YS 489 and pH 4.8 for T. saccharolyticum B6A-RI), and slightly different thermostabilities (stable for 1 h at 80 and 85{degrees}C, respectively).
- OSTI ID:
- 625678
- Journal Information:
- Journal of Bacteriology, Journal Name: Journal of Bacteriology Journal Issue: 20 Vol. 178; ISSN JOBAAY; ISSN 0021-9193
- Country of Publication:
- United States
- Language:
- English
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