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.
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References
Danno, G. (1970),Agric. Biol. Chem. 34, 1805–1811.
Tasaki, Y., Kosugi, Y., and Kanbayashi, A. (1969),Agric. Biol. Chem. 33, 1527–1535.
Yamanaka, K. (1963),Agric. Biol. Chem. 27, 271–279.
Szmant, H. H. (1986), InIndustrial Utilization of Renewable Resources: An Introduction. Technomic, Lancaster, PA.
Sipos, T. (1973), U.S. Patent 3,708,387.
Strandberg, G. W. and Smiley, K. L. (1971),Appl. Microbiol. 21, 588–593.
Posno, M., Heuvelmans, P. T. H. M, van Giezen, M. J. F., Lokman, B. C, Leer, R. J., and Pouwels, P. H. (1991),Appl. Environ. Microbiol. 57, 2764–2766.
Utt, E. A., Eddy, C. K., Keshav, K. F., and Ingram, L. O. (1991),Appl. Environ. Microbiol. 57, 1227–1234.
Schellenberg, G. D., Sarthy, A., Larson, A. E., Backer, M. P., Crabb, J. W., Lidstrom, M., Hall, B. D., and Furlong, C. E. (1984),J. Biol. Chem. 259, 6826–6832.
Wilhelm, M. and Hollenberg, C. P. (1985),Nucleic Acids Res. 13, 5717–5722.
Saari, G. C, Kumar, A. A., Kawasaki, G. H., Insley, M. Y., and O’Hara, P. J. (1987),J. Bacteriol. 169, 612–618.
Amore, R. and Hollenberg, C. P. (1989),Nucleic Acids Res. 17, 7515.
Dekker, K. A., Yamagata, H., Sakaguchi, K., and Udaka, S. (1991),Agric. Biol. Chem. 55, 221–227.
Lee, C, Meng, M., Bagdasarian, M., and Zeikus, J. G. (1990),J. Biol. Chem. 265, 19, 082–19,090.
Dekker, K., Yomagata, H., Sakaguchi, K., and Udaka, S. (1991),J. Bacteriol. 173, 3078–3083.
Rangarajan, M. and Hartely, B. S. (1992),Biochem. J. 283, 223–233.
Drocourt, D., Bejar, S., Calmels, T., Reynes, J. P., and Tiraby, G. (1988),Nucleic Acids Res. 16, 9337.
Kikuchi, T., Itoh, Y., Kasumi, T., Fukazawa, C. (1990),Agric. Biol. Chem. 54, 2469–2472.
Feldmann, S. D., Hremann, S., and Sprenger, G. A. (1992),Mol. Gen. Genet. 234, 201–210.
Bor, -Y. -C, Moraes, C, Lee, -S.-P., Crosby, W. L., Sinsky, A. J., and Batt, C. A. (1992),Gene 114, 127–131.
Chan, -E. -C, Ueng, P. P., and Chen, L. F. (1989),Appl. Microbiol. Biotechnol. 31, 524–528.
Deng, X. X. and Ho, N. W. Y. (1990),Appl. Biochem. Biotechnol. 24/25, 193–199.
Carrel, H. L., Gulsker, J. P., Burger, V., Manfre, F., Tritsch, D. and Biellman, J. P. (1989),Ptroc. Natl. Acad. Sci. USA,86, 4440.
Collyer, C. A., Henrick, K., and Blow, D. M. (1990),J. Mol. Biol. 212, 211–235.
Lehmacher, A. and Biswanger, H. (1990),J. Gen. Microbiol. 136, 679–686.
Jensen, V. J. and Rugh, S. (1987),Methods Enzymol. 136, 356–370.
Volkin, D. B. and Klibanov, A. M. (1989),Biotechnol. Bioeng. 33, 1104–1111.
Dekker, K., Sugiura, A., Yomagata, H., Sakaguchi, K., and Udaka, S. (1992),Appl. Microbiol. Biotechnol. 36, 727–732.
Merino, E., Balbas, P., Puente, J. L., and Bolivar, F. (1994),Nucleic Acids Res. 22, 1903–1908.
Merino, E., Balbas, P., and Bolivar, F. (1991),Orig. Life. Evol. Biosph. 21, 251–254.
Kumamoto, C. A. and Nault, A. K. (1989),Gene 75, 167–175.
Miller, J. H. (1972), inExperiments in Molecular Genet. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Bradford, M. M. (1976),Anal. Biochem. 72, 248–254.
Laemmli, U. K. (1970),Nature 227, 680–685.
Sambrook, J., Fritch, E., and Maniatis, T. (1989), inMolecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Hanahan, D. (1983),J. Mol. Biol. 53, 159–162.
Bragger, J. M., Daniel, R. M., Coolbear, T., and Morgan, H. W. (1989),Appl. Microbiol. Biotechnol. 31, 556–561.
Novo-Nordisk. (1994), Patent WO9207069.
Vieille, C, Hess, J. M., Kelly, R. M., and Zeikus, J. G. (1995),Appl. Environ. Microbiol. 61, 1867–1875.
Chen, W.-P. (1980),Process Biochem. Aug–Sep., 30–35.
Boyer, H. W. and Roulland-Dussiox, D. (1969),J. Mol. Biol. 41, 459–472.
Amann, E. and Brosius, J. (1985),Gene 40, 183–190.
Oshima, T. and Imahori, K. (1971),J. Gen. Microbiol. 17, 513–517.
Vieira, J. and Messing J. (1982),Gene 19, 259–268.
Nash, J. (1991), PrimerGen Program Ver. 1.1, National Research Council of Canada.
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Park, B.C., Koh, S., Chang, C. et al. Cloning and expression of the gene for xylose isomerase fromThermus flavus AT62 inEscherichia coli . Appl Biochem Biotechnol 62, 15–27 (1997). https://doi.org/10.1007/BF02787980
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DOI: https://doi.org/10.1007/BF02787980