Applied Biochemistry and Biotechnology

, Volume 62, Issue 1, pp 15–27

Cloning and expression of the gene for xylose isomerase fromThermus flavus AT62 inEscherichia coli

  • Byoung Chul Park
  • Sukhoon Koh
  • Changsoo Chang
  • Se Won Suh
  • Dae-Sil Lee
  • Si Myung Byun
Original Articles

DOI: 10.1007/BF02787980

Cite this article as:
Park, B.C., Koh, S., Chang, C. et al. Appl Biochem Biotechnol (1997) 62: 15. doi:10.1007/BF02787980

Abstract

The gene encoding xylose isomerase (xylA) was cloned fromThermus flavus AT62 and the DNA sequence was determined. ThexylA gene encodes the enzyme xylose isomerase (XI orxylA) consisting of 387 amino acids (calculated Mr of 44,941). Also, there was a partial xylulose kinase gene that was 4 bp overlapped in the end of XI gene. The XI gene was stably expressed inE. coli under the control oftac promoter. XI produced inE. coli was simply purified by heat treatment at 90°C for 10 min and column chromatography of DEAE-Sephacel. The Mr of the purified enzyme was estimated to be 45 kDa on SDS-polyacrylamide gel electrophoresis. However, Mr of the cloned XI was 185 kDa on native condition, indicating that the XI consists of homomeric tetramer. The enzyme has an optimum temperature at 90°C. Thermostability tests revealed that half life at 85°C was 2 mo and 2 h at 95°C. The optimum pH is around 7.0, close to where by-product formation is minimal. The isomerization yield of the cloned XI was about 55% from glucose, indicating that the yield is higher than those of reported enzymes. The Km values for various sugar substrates were calculated as 106 mM for glucose. Divalent cations such as Mn2+, Co2+, and Mg2+ are required for the enzyme activity and 100 mM EDTA completely inhibited the enzyme activity.

Index entries

Thermusflavus AT62 xylose isomerase xylulose kinase overlapping gene gene expression tac promoter 

Copyright information

© Springer-Verlag 1997

Authors and Affiliations

  • Byoung Chul Park
    • 1
    • 2
  • Sukhoon Koh
    • 1
  • Changsoo Chang
    • 1
    • 3
  • Se Won Suh
    • 1
    • 3
  • Dae-Sil Lee
    • 1
  • Si Myung Byun
    • 2
    • 4
  1. 1.Korea Research Institute of Bioscience and BiotechnologyYusongKorea
  2. 2.Department of Biological ScienceKorea Advanced Institute of Science and TechnologyYusongKorea
  3. 3.Department of ChemistrySeoul National UniversitySeoulKorea
  4. 4.Research Center for New Bio-Materials in AgricultureSeoul National UniversitySuwonKorea

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