1887

Microbiology Society logo

Volume 135, Issue 11

Physiology of Amidase Production by Isolation of Hyperactive Strains Using Continuous Culture Free

Abstract

The obligately methylotrophic bacterium hydrolyses aliphatic amides to ammonia and aliphatic acid using a cytoplasmic amidase. Physiological regulation of amidase activity was investigated by growing the organism under various conditions in batch, fed-batch and continuous culture. The results showed that synthesis of the enzyme was induced by various amides (acrylamide > acetamide) and repressed by ammonia. Growth of the wild-type organism in acetamide-limited continuous culture at very low dilution rate ( 0·025 h) led to the selection of a hyperactive strain (MM6), the subsequent growth of which under acrylamide limitation led to the selection of another strain (MM8) which showed even higher activity. The amidase activities of strains MM6 and MM8 were respectively approximately four and twelve times higher than that of the wild-type organism following growth under similar conditions, whereas the concentrations of the enzyme as determined by SDS-PAGE and scanning densitometry were approximately four times higher than the wild-type organism in both strains. The amidase in strain MM8 exhibited a for acrylamide that was approximately one-third lower than that of the wild-type organism or of strain MM6. It is concluded that the hyperactivity of strain MM6 was due predominantly to the production of more wild-type enzyme, whilst the hyperactivity of strain MM8 was due to the production of approximately the same amount of enzyme as strain MM6 (up to 25% of the total cell protein depending on the nature of the limiting amide) but with a substantially enhanced catalytic activity ( ). These changes were apparently the result of spontaneous mutations that occurred in response to growth at extremely low amide concentrations, giving the novel strains a strong selective advantage under these conditions (possibly by enhancing the rate of diffusion of amide into the cell).

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
© Society for General Microbiology 1989
Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-135-11-3153
1989-11-01
2024-10-03
Loading full text...

Full text loading...

/deliver/fulltext/micro/135/11/mic-135-11-3153.html?itemId=/content/journal/micro/10.1099/00221287-135-11-3153&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 the Methylo- trophs. London:: Academic Press.;
     [Google Scholar]
  3. Asano Y., Tachibana M., Tani Y., Yamada H. 1982; Purification and characterisation of amidase which participates in nitrile degradation.. Agricultural Biological Chemistry 46:1175–1181
     [Google Scholar]
  4. Beardsmore A. J., Aperghis P.N.G., Quayle J. R. 1982; Characterization of the assimilatory and dissimilatory pathways of carbon metabolism during growth of Methylophilus methylotrophus on methanol.. European Journal of Biochemistry 138:611–615
     [Google Scholar]
  5. 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]
  6. Burton S. M., Byrom D., Carver M. A., Jones G.D.D., Jones C. W. 1983; The oxidation of methylated amines by the methylotrophic bacterium Methylophilus methylotrophus.. FEMS Microbiology Letters 17:185–190
     [Google Scholar]
  7. Cairns J., Overbaugh J., Miller S. 1988; The origin of mutants.. Nature London: 335:142–145
     [Google Scholar]
  8. Clarke P. H. 1970; The aliphatic amidases of Pseudomonas aeruginosa.. Advances in Microbial Physiology 4:179–222
     [Google Scholar]
  9. Clarke P. H. 1984; Amidases of Pseudomonas aeruginosa.. In Microorganisms as Model Systems for Studying Evolution, pp 187–231 Mortlock R. P. Edited by New York: Plenum Press;
     [Google Scholar]
  10. Clarke P. H., Drew R. 1988; An experiment in enzyme evolution. Studies with Pseudomonas aeruginosa amidase.. Bioscience Reviews 8:103–120
     [Google Scholar]
  11. Cornish A., Greenwood J. A., Jones C. W. 1988a; Binding-protein-dependent glucose transport by Agrobacterium radiobacter grown in glucose- limited continuous culture.. Journal of General Microbiology 134:3099–3110
     [Google Scholar]
  12. Cornish A., Greenwood J. A., Jones C. W. 1988b; The relationship between glucose transport and the production of succinoglucan exopolysaccharide by Agrobacterium radiobacter.. Journal of General Microbiology 1343111–3122
     [Google Scholar]
  13. Dykhuizen D. E., Hartl D. L. 1983; Selection in chemostats.. Microbiology Reviews 47:150–168
     [Google Scholar]
  14. Dykhuizen D. E., Dean A. M., Hartl D. L. 1987; Metabolic flux and fitness.. Genetics 115:25–31
     [Google Scholar]
  15. Fersht A. R. 1985 Enzyme Structure and Mechanism,, 2nd edn.. New York:: W. H. Freeman.;
     [Google Scholar]
  16. Friedrich C. G., Mitrenga G. 1981; Utilization of aliphatic amides and formation of two different amidases by Alcaligenes eutrophus.. Journal of General Microbiology 125:367–374
     [Google Scholar]
  17. Greenwood J. A., Jones C. W. 1986; Environmental regulation of the methanol oxidase system of Methylophilus methylotrophus.. Journal of General Microbiology 132:1247–1256
     [Google Scholar]
  18. Hames B. D. 1981; An introduction to polyacrylamide gel electrophoresis.. In Gel Electrophoresis of Proteins; A Practical Approach, pp 1–91 Hames B. D., Rickwood D. Edited by Oxford: IRL Press;
     [Google Scholar]
  19. Harder W., Dijkhuizen L. 1983; Physiological responses to nutrient limitation.. Annual Review of Microbiology 37:1–23
     [Google Scholar]
  20. Harder W., Kuenen J. G., Matin A. 1977; Microbial selection in continuous culture.. Journal of Applied Bacteriology 43:1–24
     [Google Scholar]
  21. Hartley B. S. 1984; Experimental evolution of ribitol dehydrogenase.. In Microorganisms as Model Systems for Studying Evolution, pp 23–54 Mortlock R. P. Edited by New York: Plenum Press;
     [Google Scholar]
  22. Jones C. W., Greenwood J. A., Burton S. M., Santos H., Turner D. L. 1987; Environmental regulation of methanol and formaldehyde metabolism by Methylophilus methylotrophus.. Journal of General Microbiology 133:1511–1519
     [Google Scholar]
  23. Kubitschek H. E. 1974; Operation of selection pressure on microbial populations.. Symposia of the Society for General Microbiology 24:105–130
     [Google Scholar]
  24. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4.. Nature London: 227:680–685
     [Google Scholar]
  25. Maestracci M., Thiery A., Bui K., Arnaud A., Galzy P. 1984; Activity and regulation of an amidase (acylamide amidohydrolase EC 3.5.1.4) with a wide substrate spectrum from a Brevibacterium sp.. Archives of Microbiology 138:315–320
     [Google Scholar]
  26. Maestracci M., Thiery A., Arnaud A., Galzy P. 1986; A study of the mechanism of the reactions catalysed by the amidase from Brevibacterium sp. R312.. Agricultural and Biological Chemistry 50:2237–2241
     [Google Scholar]
  27. Miller J. M., Knowles C. J. 1984; The cellular location of nitrilase and amidase enzymes of Brevibacterium sp. R312.. FEMS Microbiology Letters 21:147–151
     [Google Scholar]
  28. Muftic M. K. 1964; A new phenol-hypochlorite reaction for ammonia.. Nature London: 201:622–623
     [Google Scholar]
  29. Neijssel O. M., Heuting S., Crabbendam K. J., Tempest D. W. 1975; Dual pathways of glycerol assimilation in Klebsiella aerogenes NCIB 418; their regulation and possible functional significance.. Archives of Microbiology 104:83–87
     [Google Scholar]
  30. Neu H. C., Heppel L. A. 1965; The release of enzymes from Escherichia coli by osmotic shock and during the formation of sphaeroplasts.. Journal of Biological Chemistry 240:3685–3692
     [Google Scholar]
  31. Novik A., Szilard L. 1950; Experiments with the chemostat on spontaneous mutations of bacteria.. Proceedings of the National Academy of Sciences of the United States of America 36:708–719
     [Google Scholar]
  32. Shaw W. V. 1987; Protein engineering; the design, synthesis and characterization of factitious proteins.. Biochemical Journal 246:1–17
     [Google Scholar]
  33. Thalenfeld B., Grossowicz N. 1976; Regulatory properties of an inducible aliphatic amidase in a thermophilic bacillus.. Journal of General Microbiology 94:131–141
     [Google Scholar]
  34. Thiery A., Maestracci M., Arnaud A., Galzy P. 1986a; Acyltransferase activity of the wide spectrum amidase of Brevibacterium sp. R312.. Journal of General Microbiology 132:2205–2208
     [Google Scholar]
  35. Thiery A., Maestracci M., Arnaud A., Galzy P., Nicolas M. 1986b; Purification and properties of an acylamide amidohydrolase (EC 3.5.1.4) with a wide activity spectrum from Brevibacterium sp. R312.. Journal of Basic Microbiology 5:299–311
     [Google Scholar]
  36. Tsang E.W.T., Grootwassink J.W.D. 1988; Extraordinarily rapid appearance of a β-fructofuran- osidase (exo-inulase) hyperproducing mutant in continuous culture of Kluyveromyces fragilis.. Journal of General Microbiology 134:679–688
     [Google Scholar]
  37. Vasey R. B., Powell K. A. 1984; Single-cell protein.. Biotechnology and Genetic Engineering Reviews 2:285–310
     [Google Scholar]
  38. Weber K., Osborne M. 1975; Proteins and sodium dodecyl sulphate: molecular weight determinations on polyacrylamide gels and related procedures.. In The Proteins I,, 3rd. pp 179–223 Neurath H., Hill R. L. Edited by London: Academic Press;
     [Google Scholar]
  39. Windass J. D., Worsey M. J., Ploli 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]
  40. Wyndham R. C., Slater H. J. 1986; A comparative study of acquired amidase activity in Pseudomonas species.. Journal of General Microbiology 132:2195–2204
     [Google Scholar]
/content/journal/micro/10.1099/00221287-135-11-3153
Loading
Physiology of Amidase Production by Methylophilus methylotrophus: Isolation of Hyperactive Strains Using Continuous Culture
Microbiology 135, 3153 (1989); https://doi.org/10.1099/00221287-135-11-3153
/content/journal/micro/10.1099/00221287-135-11-3153
/content/journal/micro/10.1099/00221287-135-11-3153
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