Skip to main content
PMC home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1990 Sep;56(9):2677–2683. doi: 10.1128/aem.56.9.2677-2683.1990

Xylanase from the extremely thermophilic bacterium "Caldocellum saccharolyticum": overexpression of the gene in Escherichia coli and characterization of the gene product.

E Lüthi 1, N B Jasmat 1, P L Bergquist 1
PMCID: PMC184827  PMID: 2275529

Abstract

A xylanase encoded by the xynA gene of the extreme thermophile "Caldocellum saccharolyticum" was overexpressed in Escherichia coli by cloning the gene downstream from the temperature-inducible lambda pR and pL promoters of the expression vector pJLA602. Induction of up to 55 times was obtained by growing the cells at 42 degrees C, and the xylanase made up to 20% of the whole-cell protein content. The enzyme was located in the cytoplasmic fraction in E. coli. The temperature and pH optima were determined to be 70 degrees C and pH 5.5 to 6, respectively. The xylanase was stable for at least 72 h if incubated at 60 degrees C, with half-lives of 8 to 9 h at 70 degrees C and 2 to 3 min at 80 degrees C. The enzyme had high activity on xylan and ortho-nitrophenyl beta-D-xylopyranoside and some activity on carboxymethyl cellulose and para-nitrophenyl beta-D-cellobioside. The gene was probably expressed from its own promoter in E. coli. Translation of the xylanase overproduced in E. coli seemed to initiate at a GTG codon and not at an ATG codon as previously determined.

Full text

PDF
2677

Images in this article

2680Image
on p.2680
2680Image
on p.2680

Selected References

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

  1. Bergquist P. L., Love D. R., Croft J. E., Streiff M. B., Daniel R. M., Morgan W. H. Genetics and potential biotechnological applications of thermophilic and extremely thermophilic micro-organisms. Biotechnol Genet Eng Rev. 1987;5:199–244. doi: 10.1080/02648725.1987.10647838. [DOI] [PubMed] [Google Scholar]
  2. Croft J. E., Love D. R., Bergquist P. L. Expression of leucine genes from an extremely thermophilic bacterium in Escherichia coli. Mol Gen Genet. 1987 Dec;210(3):490–497. doi: 10.1007/BF00327202. [DOI] [PubMed] [Google Scholar]
  3. Finlay B. B., Pasloske B. L., Paranchych W. Expression of the Pseudomonas aeruginosa PAK pilin gene in Escherichia coli. J Bacteriol. 1986 Feb;165(2):625–630. doi: 10.1128/jb.165.2.625-630.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Ghangas G. S., Hu Y. J., Wilson D. B. Cloning of a Thermomonospora fusca xylanase gene and its expression in Escherichia coli and Streptomyces lividans. J Bacteriol. 1989 Jun;171(6):2963–2969. doi: 10.1128/jb.171.6.2963-2969.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Gilbert H. J., Sullivan D. A., Jenkins G., Kellett L. E., Minton N. P., Hall J. Molecular cloning of multiple xylanase genes from Pseudomonas fluorescens subsp. cellulosa. J Gen Microbiol. 1988 Dec;134(12):3239–3247. doi: 10.1099/00221287-134-12-3239. [DOI] [PubMed] [Google Scholar]
  6. Grépinet O., Chebrou M. C., Béguin P. Purification of Clostridium thermocellum xylanase Z expressed in Escherichia coli and identification of the corresponding product in the culture medium of C. thermocellum. J Bacteriol. 1988 Oct;170(10):4576–4581. doi: 10.1128/jb.170.10.4576-4581.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hennecke H., Günther I., Binder F. A novel cloning vector for the direct selection of recombinant DNA in E. coli. Gene. 1982 Sep;19(2):231–234. doi: 10.1016/0378-1119(82)90011-7. [DOI] [PubMed] [Google Scholar]
  8. Johnson D. A., Willetts N. S. Construction and characterization of multicopy plasmids containing the entire F transfer region. Plasmid. 1980 Nov;4(3):292–304. doi: 10.1016/0147-619x(80)90068-2. [DOI] [PubMed] [Google Scholar]
  9. Kudo T., Ohkoshi A., Horikoshi K. Molecular cloning and expression of a xylanase gene of alkalophilic Aeromonas sp. no. 212 in Escherichia coli. J Gen Microbiol. 1985 Oct;131(10):2825–2830. doi: 10.1099/00221287-131-10-2825. [DOI] [PubMed] [Google Scholar]
  10. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  11. 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]
  12. Lever M. Colorimetric and fluorometric carbohydrate determination with p-hydroxybenzoic acid hydrazide. Biochem Med. 1973 Apr;7(2):274–281. doi: 10.1016/0006-2944(73)90083-5. [DOI] [PubMed] [Google Scholar]
  13. Love D. R., Fisher R., Bergquist P. L. Sequence structure and expression of a cloned beta-glucosidase gene from an extreme thermophile. Mol Gen Genet. 1988 Jul;213(1):84–92. doi: 10.1007/BF00333402. [DOI] [PubMed] [Google Scholar]
  14. Lüthi E., Love D. R., McAnulty J., Wallace C., Caughey P. A., Saul D., Bergquist P. L. Cloning, sequence analysis, and expression of genes encoding xylan-degrading enzymes from the thermophile "Caldocellum saccharolyticum". Appl Environ Microbiol. 1990 Apr;56(4):1017–1024. doi: 10.1128/aem.56.4.1017-1024.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Matsudaira P. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem. 1987 Jul 25;262(21):10035–10038. [PubMed] [Google Scholar]
  16. Mott J. E., Grant R. A., Ho Y. S., Platt T. Maximizing gene expression from plasmid vectors containing the lambda PL promoter: strategies for overproducing transcription termination factor rho. Proc Natl Acad Sci U S A. 1985 Jan;82(1):88–92. doi: 10.1073/pnas.82.1.88. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Mountfort D. O., Asher R. A. Production of xylanase by the ruminal anaerobic fungus Neocallimastix frontalis. Appl Environ Microbiol. 1989 Apr;55(4):1016–1022. doi: 10.1128/aem.55.4.1016-1022.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. O'Neill G. P., Kilburn D. G., Warren R. A., Miller R. C., Jr Overproduction from a cellulase gene with a high guanosine-plus-cytosine content in Escherichia coli. Appl Environ Microbiol. 1986 Oct;52(4):737–743. doi: 10.1128/aem.52.4.737-743.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Panbangred W., Kondo T., Negoro S., Shinmyo A., Okada H. Molecular cloning of the genes for xylan degradation of Bacillus pumilus and their expression in Escherichia coli. Mol Gen Genet. 1983;192(3):335–341. doi: 10.1007/BF00392172. [DOI] [PubMed] [Google Scholar]
  20. Remaut E., Tsao H., Fiers W. Improved plasmid vectors with a thermoinducible expression and temperature-regulated runaway replication. Gene. 1983 Apr;22(1):103–113. doi: 10.1016/0378-1119(83)90069-0. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Schauder B., Blöcker H., Frank R., McCarthy J. E. Inducible expression vectors incorporating the Escherichia coli atpE translational initiation region. Gene. 1987;52(2-3):279–283. doi: 10.1016/0378-1119(87)90054-0. [DOI] [PubMed] [Google Scholar]
  23. Shinedling S., Gayle M., Pribnow D., Gold L. Mutations affecting translation of the bacteriophage T4 rIIB gene cloned in Escherichia coli. Mol Gen Genet. 1987 May;207(2-3):224–232. doi: 10.1007/BF00331582. [DOI] [PubMed] [Google Scholar]
  24. Short J. M., Fernandez J. M., Sorge J. A., Huse W. D. Lambda ZAP: a bacteriophage lambda expression vector with in vivo excision properties. Nucleic Acids Res. 1988 Aug 11;16(15):7583–7600. doi: 10.1093/nar/16.15.7583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sipat A., Taylor K. A., Lo R. Y., Forsberg C. W., Krell P. J. Molecular cloning of a xylanase gene from Bacteroides succinogenes and its expression in Escherichia coli. Appl Environ Microbiol. 1987 Mar;53(3):477–481. doi: 10.1128/aem.53.3.477-481.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Vander Wauven C., Piérard A., Kley-Raymann M., Haas D. Pseudomonas aeruginosa mutants affected in anaerobic growth on arginine: evidence for a four-gene cluster encoding the arginine deiminase pathway. J Bacteriol. 1984 Dec;160(3):928–934. doi: 10.1128/jb.160.3.928-934.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Wain-Hobson S., Sonigo P., Danos O., Cole S., Alizon M. Nucleotide sequence of the AIDS virus, LAV. Cell. 1985 Jan;40(1):9–17. doi: 10.1016/0092-8674(85)90303-4. [DOI] [PubMed] [Google Scholar]
  28. Whitehead T. R., Hespell R. B. Cloning and expression in Escherichia coli of a xylanase gene from Bacteroides ruminicola 23. Appl Environ Microbiol. 1989 Apr;55(4):893–896. doi: 10.1128/aem.55.4.893-896.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Wong K. K., Tan L. U., Saddler J. N. Multiplicity of beta-1,4-xylanase in microorganisms: functions and applications. Microbiol Rev. 1988 Sep;52(3):305–317. doi: 10.1128/mr.52.3.305-317.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Yang R. C., MacKenzie C. R., Bilous D., Narang S. A. Hyperexpression of a Bacillus circulans xylanase gene in Escherichia coli and characterization of the gene product. Appl Environ Microbiol. 1989 May;55(5):1192–1195. doi: 10.1128/aem.55.5.1192-1195.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Yang R. C., Mackenzie C. R., Bilous D., Seligy V. L., Narang S. A. Molecular Cloning and Expression of a Xylanase Gene from Bacillus polymyxa in Escherichia coli. Appl Environ Microbiol. 1988 Apr;54(4):1023–1029. doi: 10.1128/aem.54.4.1023-1029.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES