1887

Abstract

The obligately methylotrophic bacterium methylotrophus hydrolyses acetamide and acrylamide using a cytoplasmic amidase. In previous work, continuous culture was used to isolate spontaneous mutants which overexpressed either the wild-type amidase (strain MM6) or a mutant amidase with an apparently higher (strain MM8). We now report that NTG mutagenesis of strain MM8 followed by acrylamide-limited growth at low dilution rate ( 0·025 h; 37 °C) led to the selection of a strain which continued to overexpress the amidase, but which exhibited an unexpectedly low amidase activity and a greatly decreased for acrylamide (strain MM15). Amidases from the wild-type and mutant strains were purified and shown to be homotetramers (subunit 38000, pI 4·1). The N-terminal amino acid sequence of the wild-type enzyme was 90% homologous with the aliphatic amidase from , and Southern blotting using an oligonucleotide probe for this region showed that overexpression of the enzyme in the mutant strains was not due to gene amplification. Compared with the wild-type and MM6 enzymes, the MM8 enzyme exhibited a threefold higher and a slightly lower for acrylamide, whereas the MM15 enzyme exhibited a similar and an eightfold lower for acrylamide. The MM15 enzyme also reacted more extensively with the thiol group reagent DTNB, had a significantly lower sedimentation coefficient and exhibited a more relaxed substrate specificity, all of which were compatible with a looser tetrameric structure. It was also much more susceptible than the other three enzymes to inactivation by high temperature or by freezing and thawing (MM15»MM8>MM6/wild-type), both of which variably dissociated the enzyme into inactive dimers and monomers. The amidase activity of strain MM15 was almost 15-fold higher following growth at 25 °C than at 37 °C, since at this lower temperature the enzyme exhibited a similar to the MM8 enzyme and was not significantly dissociated. However, as strain MM15 readily outgrew the organism from which it was derived (strain MM8) during acrylamide-limited continuous culture at 37 °C, it is clear that under these conditions a low was a greater selective advantage than a high .

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-137-1-169
1991-01-01
2021-04-20
Loading full text...

Full text loading...

/deliver/fulltext/micro/137/1/mic-137-1-169.html?itemId=/content/journal/micro/10.1099/00221287-137-1-169&mimeType=html&fmt=ahah

References

  1. Ambler R. P., Auffret A. D., Clarke P. H. 1987; The amino acid sequence of the aliphatic amidase from Pseudomonas aeruginosa . FEBS Letters 215:285–290
    [Google Scholar]
  2. Anthony C. 1982 The Biochemistry of Methylotrophs London: Academic Press;
    [Google Scholar]
  3. Asano Y., Tachibana M., Tani Y., Yamada H. 1982; Purification and characterization of amidase which participates in nitrile degradation. Agricultural Biological Chemistry 46:1175–1181
    [Google Scholar]
  4. Brammar W. J., Charles I. G., Matfield M., Cheng-Pin L., Drew R. E., Clarke P. H. 1987; The nucleotide sequence of the amiE gene of Pseudomonas aeruginosa . FEBS Letters 215:291–294
    [Google Scholar]
  5. Chow L. T., Kahmann R., Kamp D. 1977; Electron microscopic characterization of DNAs of non-defective deletion mutants of bacteriophage Mu. Journal of Molecular Biology 113:591–609
    [Google Scholar]
  6. Clarke P. H. 1970; The aliphatic amidases of Pseudomonas aeruginosa . Advances in Microbial Physiology 4:179–222
    [Google Scholar]
  7. Clarke P. H. 1984; Amidases of Pseudomonas aeruginosa . Microorganisms as Model Systems for Studying Evolution187–231 Mortlock R. P. New York: Plenum Press;
    [Google Scholar]
  8. Clarke P. H., Drew R. 1988; An experiment in enzyme evolution. Studies with Pseudomonas aeruginosa amidase. Bioscience Reviews 8:103–120
    [Google Scholar]
  9. Dykhuizen D. E., Hartl D. L. 1983; Selection in chemostats. Microbiological Reviews 47:150–168
    [Google Scholar]
  10. Dykhuizen D. E., Dean A. M., Hartl D. L. 1987; Metabolic flux and fitness. Genetics 115:25–31
    [Google Scholar]
  11. Hames B. D. 1981; An introduction to polyacrylamide gel electrophoresis. Gel Electrophoresis of Proteins; a Practical Approach1–91 Hames B. D., Rickwood D. Oxford: IRL Press;
    [Google Scholar]
  12. Harder W., Kuenen J. G., Matin A. 1977; Microbial selection in continuous culture. Journal of Applied Bacteriology 43:1–24
    [Google Scholar]
  13. Hartley B. S. 1984; Experimental evolution of ribitol dehydro-genase. Microorganisms as Model Systems for Studying Evolution23–54 Mortlock R. P. New York: Plenum Press;
    [Google Scholar]
  14. Kubitschek H. E. 1974; Operation of selection pressure on microbial populations. Symposia of the Society for General Microbiology 24:105–130
    [Google Scholar]
  15. Large P. J., Bamforth C. W. 1988 Methylotrophy and Biotechnology London: Longman;
    [Google Scholar]
  16. Maestracci M., Thiery A., Bui K., Arnaud A., Galzy P. 1984; Activity and regulation of an amidase (acylamide amidohy-drolase EC 3.5.1.4) with a wide substrate spectrum from a Brevibacterium sp. Archives of Microbiology 138:315–320
    [Google Scholar]
  17. Maestracci M., Bui K., Thiery A., Arnaud A., Galzy P. 1988; The amidases from a Brevibacterium strain: study and applications. Advances in Biochemical Engineering/Biotechnology 36:67–115
    [Google Scholar]
  18. Maniatis T., Fritsch E. F., Sambrook J. 1982 Molecular Cloning: a Laboratory Manual Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  19. Mortlock R. P. 1982; Metabolic acquisitions through laboratory selection. Annual Review of Microbiology 36:259–284
    [Google Scholar]
  20. Rowe A. J. 1984; Techniques for determining molecular weight. Techniques in the Life Sciences Bl/1BS106 Tipton K. F. Amsterdam: Elsevier;
    [Google Scholar]
  21. Silman N. J., Carver M. A., Jones C. W. 1989; Physiology of amidase production by Methylophilus methylotrophus: isolation of hyperactive strains using continuous culture. Journal of General Microbiology 135:3153–3164
    [Google Scholar]
  22. Spragg S. P. 1980 The Physical Behaviour of Macromolecules with Biological Functions66–68 Chichester & New York: Wiley;
    [Google Scholar]
  23. Thiery A., Maestracci M., Arnaud A., Galzy P., Nicolas M. 1986; Purification and properties of an acylamide amidohydrolase (EC 3.5.1.4) with a wide variety spectrum from Brevibacterium sp. R312. Journal of Basic Microbiology 5:299–311
    [Google Scholar]
  24. Vasey R. B., Powell K. A. 1984; Single-cell protein. Biotechnology and Genetic Engineering Reviews 2:285–310
    [Google Scholar]
  25. Windass J. D., Worsey M. J., Pioli E. M., Barth P. T., Atherton K. T., Dart E. C, Byrom D., Powell K., Senior P. J. 1980; Improved conversion of methanol to single cell protein by Methylophilus methylotrophus . Nature, London 287:396–401
    [Google Scholar]
  26. Wyndham R. C., Slater H. J. 1986; A comparative study of acquired amidase activity in Pseudomonas sp. Journal of General Microbiology 132:2195–2204
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-137-1-169
Loading
/content/journal/micro/10.1099/00221287-137-1-169
Loading

Data & Media loading...

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