Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1996 Oct;178(20):5938–5945. doi: 10.1128/jb.178.20.5938-5945.1996

Purification and cloning of a thermostable xylose (glucose) isomerase with an acidic pH optimum from Thermoanaerobacterium strain JW/SL-YS 489.

S Y Liu 1, J Wiegel 1, F C Gherardini 1
PMCID: PMC178450  PMID: 8830690

Abstract

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 C) 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 Mn2+, Co2+, or Mg2+ 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 the 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 isomerases in the database showed that the enzyme had 98% homology with a xylose isomerase from a closely 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 4.7 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). The obvious difference was the pH optimum (6.4 to 6.8 and 7.0 to 7.5, respectively). The fact that the pH optimum of the enzyme from strain JW/SL-YS 489 was the property that differed significantly from the T. saccharolyticum B6A-RI xylose isomerase suggested that one or more of the observed amino acid changes was responsible for this observed difference.

Full Text

The Full Text of this article is available as a PDF (945.3 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Amore R., Hollenberg C. P. Xylose isomerase from Actinoplanes missouriensis: primary structure of the gene and the protein. Nucleic Acids Res. 1989 Sep 25;17(18):7515–7515. doi: 10.1093/nar/17.18.7515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bor Y. C., Moraes C., Lee S. P., Crosby W. L., Sinskey A. J., Batt C. A. Cloning and sequencing the Lactobacillus brevis gene encoding xylose isomerase. Gene. 1992 May 1;114(1):127–132. doi: 10.1016/0378-1119(92)90718-5. [DOI] [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  5. Briggs K. A., Lancashire W. E., Hartley B. S. Molecular cloning, DNA structure and expression of the Escherichia coli D-xylose isomerase. EMBO J. 1984 Mar;3(3):611–616. doi: 10.1002/j.1460-2075.1984.tb01856.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Carrell H. L., Glusker J. P., Burger V., Manfre F., Tritsch D., Biellmann J. F. X-ray analysis of D-xylose isomerase at 1.9 A: native enzyme in complex with substrate and with a mechanism-designed inactivator. Proc Natl Acad Sci U S A. 1989 Jun;86(12):4440–4444. doi: 10.1073/pnas.86.12.4440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. DISCHE Z., BORENFREUND E. A new spectrophotometric method for the detection and determination of keto sugars and trioses. J Biol Chem. 1951 Oct;192(2):583–587. [PubMed] [Google Scholar]
  8. Dekker K., Yamagata H., Sakaguchi K., Udaka S. Xylose (glucose) isomerase gene from the thermophile Thermus thermophilus: cloning, sequencing, and comparison with other thermostable xylose isomerases. J Bacteriol. 1991 May;173(10):3078–3083. doi: 10.1128/jb.173.10.3078-3083.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Drocourt D., Bejar S., Calmels T., Reynes J. P., Tiraby G. Nucleotide sequence of the xylose isomerase gene from Streptomyces violaceoniger. Nucleic Acids Res. 1988 Oct 11;16(19):9337–9337. doi: 10.1093/nar/16.19.9337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Henrick K., Collyer C. A., Blow D. M. Structures of D-xylose isomerase from Arthrobacter strain B3728 containing the inhibitors xylitol and D-sorbitol at 2.5 A and 2.3 A resolution, respectively. J Mol Biol. 1989 Jul 5;208(1):129–157. doi: 10.1016/0022-2836(89)90092-2. [DOI] [PubMed] [Google Scholar]
  12. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  13. Lam V. M., Daruwalla K. R., Henderson P. J., Jones-Mortimer M. C. Proton-linked D-xylose transport in Escherichia coli. J Bacteriol. 1980 Jul;143(1):396–402. doi: 10.1128/jb.143.1.396-402.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lee C. Y., Bagdasarian M., Meng M. H., Zeikus J. G. Catalytic mechanism of xylose (glucose) isomerase from Clostridium thermosulfurogenes. Characterization of the structural gene and function of active site histidine. J Biol Chem. 1990 Nov 5;265(31):19082–19090. [PubMed] [Google Scholar]
  15. Lee C. Y., Bhatnagar L., Saha B. C., Lee Y. E., Takagi M., Imanaka T., Bagdasarian M., Zeikus J. G. Cloning and expression of the Clostridium thermosulfurogenes glucose isomerase gene in Escherichia coli and Bacillus subtilis. Appl Environ Microbiol. 1990 Sep;56(9):2638–2643. doi: 10.1128/aem.56.9.2638-2643.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lee Y. E., Ramesh M. V., Zeikus J. G. Cloning, sequencing and biochemical characterization of xylose isomerase from Thermoanaerobacterium saccharolyticum strain B6A-RI. J Gen Microbiol. 1993 Jun;139(Pt 6):1227–1234. doi: 10.1099/00221287-139-6-1227. [DOI] [PubMed] [Google Scholar]
  17. Meaden P. G., Aduse-Opoku J., Reizer J., Reizer A., Lanceman Y. A., Martin M. F., Mitchell W. J. The xylose isomerase-encoding gene (xylA) of Clostridium thermosaccharolyticum: cloning, sequencing and phylogeny of XylA enzymes. Gene. 1994 Apr 8;141(1):97–101. doi: 10.1016/0378-1119(94)90134-1. [DOI] [PubMed] [Google Scholar]
  18. Pohl T. Concentration of proteins and removal of solutes. Methods Enzymol. 1990;182:68–83. doi: 10.1016/0076-6879(90)82009-q. [DOI] [PubMed] [Google Scholar]
  19. Rangarajan M., Hartley B. S. Mechanism of D-fructose isomerization by Arthrobacter D-xylose isomerase. Biochem J. 1992 Apr 1;283(Pt 1):223–233. doi: 10.1042/bj2830223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Rey F., Jenkins J., Janin J., Lasters I., Alard P., Claessens M., Matthyssens G., Wodak S. Structural analysis of the 2.8 A model of Xylose isomerase from Actinoplanes missouriensis. Proteins. 1988;4(3):165–172. doi: 10.1002/prot.340040303. [DOI] [PubMed] [Google Scholar]
  21. Russell A. J., Fersht A. R. Rational modification of enzyme catalysis by engineering surface charge. Nature. 1987 Aug 6;328(6130):496–500. doi: 10.1038/328496a0. [DOI] [PubMed] [Google Scholar]
  22. Saari G. C., Kumar A. A., Kawasaki G. H., Insley M. Y., O'Hara P. J. Sequence of the Ampullariella sp. strain 3876 gene coding for xylose isomerase. J Bacteriol. 1987 Feb;169(2):612–618. doi: 10.1128/jb.169.2.612-618.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Short J. M., Sorge J. A. In vivo excision properties of bacteriophage lambda ZAP expression vectors. Methods Enzymol. 1992;216:495–508. doi: 10.1016/0076-6879(92)16045-l. [DOI] [PubMed] [Google Scholar]
  25. Siddiqui K. S., Loviny-Anderton T., Rangarajan M., Hartley B. S. Arthrobacter D-xylose isomerase: chemical modification of carboxy groups and protein engineering of pH optimum. Biochem J. 1993 Dec 15;296(Pt 3):685–691. doi: 10.1042/bj2960685. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Smith C. A., Rangarajan M., Hartley B. S. D-Xylose (D-glucose) isomerase from Arthrobacter strain N.R.R.L. B3728. Purification and properties. Biochem J. 1991 Jul 1;277(Pt 1):255–261. doi: 10.1042/bj2770255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Spomer W. E., Wootton J. F. The hydrolysis of alpha-N-benzoyl-L-argininamide catalyzed by trypsin and acetyltrypsin. Dependence on pH. Biochim Biophys Acta. 1971 Apr 14;235(1):164–171. doi: 10.1016/0005-2744(71)90044-1. [DOI] [PubMed] [Google Scholar]
  28. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  29. Valenzuela P., Bender M. L. Kinetic properties of succinylated and ethylenediamine-amidated -chymotrypsins. Biochim Biophys Acta. 1971 Dec 15;250(3):538–548. doi: 10.1016/0005-2744(71)90254-3. [DOI] [PubMed] [Google Scholar]
  30. Vieille C., Hess J. M., Kelly R. M., Zeikus J. G. xylA cloning and sequencing and biochemical characterization of xylose isomerase from Thermotoga neapolitana. Appl Environ Microbiol. 1995 May;61(5):1867–1875. doi: 10.1128/aem.61.5.1867-1875.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Whitlow M., Howard A. J., Finzel B. C., Poulos T. L., Winborne E., Gilliland G. L. A metal-mediated hydride shift mechanism for xylose isomerase based on the 1.6 A Streptomyces rubiginosus structures with xylitol and D-xylose. Proteins. 1991;9(3):153–173. doi: 10.1002/prot.340090302. [DOI] [PubMed] [Google Scholar]
  32. Wilhelm M., Hollenberg C. P. Nucleotide sequence of the Bacillus subtilis xylose isomerase gene: extensive homology between the Bacillus and Escherichia coli enzyme. Nucleic Acids Res. 1985 Aug 12;13(15):5717–5722. doi: 10.1093/nar/13.15.5717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Yamanaka K. Purification, crystallization and properties of the D-xylose isomerase from Lactobacillus brevis. Biochim Biophys Acta. 1968 Mar 25;151(3):670–680. doi: 10.1016/0005-2744(68)90015-6. [DOI] [PubMed] [Google Scholar]
  34. Zhang H., Scholl R., Browse J., Somerville C. Double stranded DNA sequencing as a choice for DNA sequencing. Nucleic Acids Res. 1988 Feb 11;16(3):1220–1220. doi: 10.1093/nar/16.3.1220. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. van Tilbeurgh H., Jenkins J., Chiadmi M., Janin J., Wodak S. J., Mrabet N. T., Lambeir A. M. Protein engineering of xylose (glucose) isomerase from Actinoplanes missouriensis. 3. Changing metal specificity and the pH profile by site-directed mutagenesis. Biochemistry. 1992 Jun 23;31(24):5467–5471. doi: 10.1021/bi00139a007. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES