1887

Microbiology Society logo

Volume 139, Issue 6

Genetic organization, sequence and biochemical characterization of recombinant -xylosidase from strain B6A-RI Free

Abstract

Endoxylanase () and -xylosidase () genes from were subcloned from a cosmid clone (pXDM1) to generate pXPH3. The nucleotide sequence of a I-III fragment in pXPH3 that contained revealed an open reading frame (ORF) of 1500 bp encoding a 55 kDa protein. Another open reading frame (ORF1) of unknown function was found 21 bp downstream from the first stop codon of . , ORF1 and had the same direction of transcription. from strain B6A-RI exhibited 45 % amino acid similarity, with 18 % amino acid identity to of strain B6A-RI, and 61 % similarity and 37 % identity with the -xylosidase gene from . Recombinant -xylosidase was purified from (pXPH3) cells. The enzyme was a monomer with a molecular mass of 55 kDa. The specific activity and pH and temperature optima for hydrolysis of -nitrophenyl---xylopyranoside (pNPX) were 5·53 U mg, 5·5 and 70 °C, respectively. The -xylosidase was stable at 65 °C, but lost activity at 85 °C. The purified enzyme had hydrolytic activity towards xylopentose, xylotriose, xylobiose and pNPX, but had no activity toward xylan.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
© Society for General Microbiology 1993
Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-139-6-1235
1993-06-01
2024-04-20
Loading full text...

Full text loading...

/deliver/fulltext/micro/139/6/mic-139-6-1235.html?itemId=/content/journal/micro/10.1099/00221287-139-6-1235&mimeType=html&fmt=ahah

References

  1. Bachmann S.L., Mccarthy A.J. 1989; Purification and characterization of a thermostable β-xylosidase from Thermomonospora fusca . Journal of General Microbiology 135:293–299
     [Google Scholar]
  2. Bernier R., Driguez H., Desrochers M. 1983; Molecular cloning of Bacillus subtilis xylanase gene in Escherichia coli . Gene 26:59–65
     [Google Scholar]
  3. Biely P. 1985; Microbial xylanolytic systems. Trends in Biotechnology . 3:286–290
     [Google Scholar]
  4. Birnboim H.C., Doly J. 1979; A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Research 7:1513–1523
     [Google Scholar]
  5. Bradford M.M. 1976; A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72:248–254
     [Google Scholar]
  6. Clewell D.B. 1972; Nature of Col El plasmid replication in Escherichia coli in the presence of chloramphenicol. Journal of Bacteriology 110:667–676
     [Google Scholar]
  7. Deleyn F., Claeyssens M., De BRUYNE C.K. 1982; β Xylosidase from Penicillium wortmanni . Methods in Enzymology 83:639–644
     [Google Scholar]
  8. Devereux J., Haeberli P., Smithies O. 1984; A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Research 12:387–395
     [Google Scholar]
  9. Hespell R.B. 1992; Fermentation of xylans by Butyrivibrio fibrisolvens and Thermoanaerobacter strain B6A: utilization of uronic acids and xylanolytic activities. Current Microbiology 25:189–195
     [Google Scholar]
  10. Hudson R.C., Schofield L.R., Coolbear T., Daniel R.M., Morgan H.W. 1991; Purification and properties of an aryl β-xylosidase from a cellulolytic extreme thermophile expressed in Escherichia coli . Biochemical Journal 273:645–650
     [Google Scholar]
  11. Kellet L.E., Poole D.M., Ferreira L.M., Durrant A.J., Hazlewood G.P., Gilbert H.J. 1990; Xylanase B and an arabinofuranosidase from Pseudomonas fluorescens subsp. cellulosa contain identical cellulose-binding domains and are encoded by adjacent genes. Biochemical Journal 272:369–376
     [Google Scholar]
  12. Kitpreechavanichi V., Hayashi M., Nagai S. 1986; Purification and characterization of extracellular β-xylosidase and β-glucosidase from Aspergillus fumigatus . Agricultural and Biological Chemistry 50:1703–1711
     [Google Scholar]
  13. Laemmli U.K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature; London: 227680–685
     [Google Scholar]
  14. Lee S.F., Forsberg C.W. 1987; Isolation and some properties of a β-d-xylosidase from Clostridium acetobutylicum ATCC 824. Applied and Environmental Microbiology 53:651–654
     [Google Scholar]
  15. Lee Y.E., Jain M.K., Lee C., Lowe S.E., Zeikus J.G. 1993a; Taxonomic distinction of saccharolytic thermoanaerobes: description of Thermoanaerobium xylanolyticum gen. nov., sp. nov. and Thermoanaerobium saccharolyticum sp. nov. Reclassification of Thermoanaerobium brockii, Clostridium thermosulfurogenes and Clostridium thermohydrosulfuricum El00-69 as Thermoanaerobacter brockii comb, nov., Thermoanaerobacterium thermosulfurigenes comb. nov. and Thermoanaerobacterium thermohydrosulfuricus comb, nov., and transfer of Clostridium thermohydrosulfuricum39E to Thermoanaerobacter ethanolicus . International Journal of Systematic Bacteriology 43:41–51
     [Google Scholar]
  16. Lee Y.-E., Lowe S.E., Zeikus J.G. 1993b; Regulation and characterization of xylanolytic enzymes of Thermoanaerobacterium saccharolyticum strain B6A-RI. Applied and Environmental Microbiology (in the Press).
    [Google Scholar]
  17. Luthi E., Love D.R., Mcanulty J., Wallace C., Caughey P.A., Saul D., Bergquist P.L. 1990; Cloning, sequence analysis, and expression of genes encoding xylan-degrading enzymes from the thermophile ‘Caldocellum saccharolyticum’. Applied and Environmental Microbiology 56:1017–1024
     [Google Scholar]
  18. Matsuo M., Yasui T. 1984; Purification and some properties of β-xylosidase from Emericella nidulans . Agricultural and Biological Chemistry 48:1853–1860
     [Google Scholar]
  19. Nanmori T., Watanabe T., Shinke R., Kohno A., Kawamura Y. 1990; Purification and properties of thermostable xylanase and β-xylosidase produced by a newly isolated Bacillus stearothermo- philus strain. Journal of Bacteriology 172:6669–6672
     [Google Scholar]
  20. Panbangred W., Kawaguchi O., Tomita T., Shinmyo A., Okada H. 1984; Isolation of two β-xylosidase genes of Bacillus pumilus and comparison of their gene products. European Journal of Biochemistry 138:267–273
     [Google Scholar]
  21. Perbal B. 1988; A Practical Guide to Molecular Cloning,2nd edn. New York: John WileySons.
    [Google Scholar]
  22. Reilly P.J. 1981; Xylanase: structure and function. Basic Life Sciences 18:111–129
     [Google Scholar]
  23. Sambrook J., Fritsch E.F., Maniatis T. 1989; Molecular Cloning: A Laboratory Manual,2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
    [Google Scholar]
  24. Sandhu J.S., Kennedy J.F. 1984; Molecular cloning of Bacillus polymixa1→ 4-β-d xylanase EC 3.2.1.8 gene in Escherichia coli . Enzyme and Microbial Technology 6:271–274
     [Google Scholar]
  25. Sanger F., Nicklen S., Coulson A.R. 1977; DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences of the United States of America 74:5463–5467
     [Google Scholar]
  26. Sewell G.W., Utt E.A., Hespell R.B., Mackenzie K.F., Ingram L.O. 1989; Identification of the Butyrivibrio fibrisolvens xylosidase gene (xylB) coding region and its expression in Escherichia coli . Applied and Environmental Microbiology 55:306–311
     [Google Scholar]
  27. Shao W., Wiegel J. 1992; Purification and characterization of a thermostable β-xylosidase from Thermoanaerobacter ethanolicus. Journal of Bacteriology . 174:5848–5853
     [Google Scholar]
  28. Whistler R. L., Richards E. L. 1970; Hemicelluloses. In The Carbohydrates - Chemistry and Biochemistry,2nd edn. 2A pp. 447–169 Pigmand W. (Edited by) Horton New York: Academic Press;
     [Google Scholar]
  29. Whitehead T.R., Hespell R.B. 1990; The genes for three xylan- degrading activities from Bacteroides ovatus are clustered in a 3-8- kilobase region. Journal of Bacteriology 172:2408–2412
     [Google Scholar]
  30. Wong K. K. Y., Tan L. U. L., Saddler J.N. 1988; Multiplicity of β1,4-xylanase in micro-organisms: functions and applications. Microbiological Reviews 52:305–317
     [Google Scholar]
  31. Xu W.-Z., Shima Y., Negoro S., Urabe I. 1991; Sequence and properties of β-xylosidase from Bacillus pumilus IPO: contradiction of the previous nucleotide sequence. European Journal of Biochemistry 202:1197–1203
     [Google Scholar]
  32. Zeikus J.G., Ben-Bassat A., Hegge P. 1980; Microbiology of methanogenesis in thermal volcanic environments. Journal of Bacteriology 143:432–440
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-139-6-1235
Loading
Genetic organization, sequence and biochemical characterization of recombinant β-xylosidase from Thermoanaerobacterium saccharolyticum strain B6A-RI
Microbiology 139, 1235 (1993); https://doi.org/10.1099/00221287-139-6-1235
/content/journal/micro/10.1099/00221287-139-6-1235
/content/journal/micro/10.1099/00221287-139-6-1235
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error