1887

Abstract

Summary: A new simple procedure for the production of protoplasts of with high regeneration rates and efficient transport activity is described, involving the use of a preparation of Novozym 234 with very low protease activity. The combination of a heat pretreatment at 55 °C for 15 min with the use of the protease inhibitor aprotinin resulted in a 97% reduction of Novozym 234 protease activity with respect to untreated controls. Polysaccharide-hydrolysing activity was inhibited much less, to 60% of the untreated Novozym 234 level. Protoplasts could be successfully produced with the new low-protease Novozym 234 preparation, showing a threefold increase in regeneration capacity compared to control protoplasts obtained with the original preparation. The rates of 3--methylglucose uptake and the capacity to accumulate this sugar analogue were also higher in protoplasts obtained by the new method.

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/content/journal/micro/10.1099/00221287-137-7-1647
1991-07-01
2021-05-09
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References

  1. Anson M. L. 1938; The estimation of pepsin, trypsin, papain and cathepsin with haemoglobin. Journal of General Physiology 22:79–89
    [Google Scholar]
  2. Beynon R. J., Salvesen G. 1989; Commercially available protease inhibitors. Proteolytic Enzymes: a Practical Approach241–249 Beynon R. J., Bond J. S. Oxford: IRL Press at Oxford University Press;
    [Google Scholar]
  3. Brock F. M., Forsberg C. W., Buchanan-Smith J. G. 1982; Proteolytic activity of rumen microorganisms and effects of proteinase inhibitors. Applied and Environmental Microbiology 44:561–569
    [Google Scholar]
  4. Collings A., Davis B., Mills J. 1988; Factors affecting protoplast release from some mesophilic, thermophilic and thermo-tolerant species of filamentous fungi using Novozym 234. Microbios 53:197–210
    [Google Scholar]
  5. Dubourdieu D., Desplanques C., Villetaz J.-C., Ribereau-Gayon P. 1985; Investigations of an industrial β-d-glucanase from Trichoderma harzianum . Carbohydrate Research 144:277–287
    [Google Scholar]
  6. Hamlyn P. F., Bradshaw R. E., Mellon F. M., Santiago C. M., Wilson J. M., Peberdy J. F. 1981; Efficient protoplast isolation from fungi using commercial enzymes. Enzyme and Microbial Technology 3:321–325
    [Google Scholar]
  7. Kitamoto Y., Mori N., Yamamoto M., Ohiwa T., Ichikawa Y. 1988; A simple method for protoplast formation and improvement of protoplast regeneration from various fungi using an enzyme from Trichoderma harzianum . Applied Microbiology and Biotechnology 28:445–450
    [Google Scholar]
  8. Leopold J., Seichertova O. 1967; The parasitism of the mould Penicilliumpurpurogenum on Aspergillus niger III. The determination of the part of some enzymes of Penicillium purpurogenum in the mycelium lysis of the mould Aspergillus niger . Folia Microbiologica 12:458–465
    [Google Scholar]
  9. Mann W., Jeffery J. 1986; Yeasts in molecular biology. Spheroplast preparation with Candida utilis, Schizosaccharomyces pombe and Saccharomyces cerevisiae . Bioscience Reports 6:597–602
    [Google Scholar]
  10. Miller G. L. 1959; Use of DNS acid reagent for determination of reducing sugar. Analytical Chemistry 31:426–428
    [Google Scholar]
  11. Peberdy J. F., Ferenczy L. 1985 Fungal Protoplasts, Applications in Biochemistry and Genetics New York: Marcel Dekker;
    [Google Scholar]
  12. Pitt D., Barnes J. C. 1987; Hexose transport during calcium-induced conidiation in Penicillium notatum . Transactions of the British Mycological Society 89:359–365
    [Google Scholar]
  13. Quigley D. R., Taft C. S., Stark T., Selitrennikoff C. P. 1987; Optimal conditions for the release of protoplasts of Neurospora using Novozym 234. Experimental Mycology 11:236–240
    [Google Scholar]
  14. Ridout C. J., Coley-Smith J. R., Lynch J. M. 1988; Fractionation of extracellular enzymes from a mycoparasitic strain of Trichoderma harzianum . Enzyme and Microbial Technology 10:180–187
    [Google Scholar]
  15. Roos W., Slavik J. 1987; Intracellular pH topography of Penicillium cyclopium protoplasts. Maintenance of ΔpH by both passive and active mechanisms. Biochimica et Biophysica Ada 899:67–75
    [Google Scholar]
  16. Sarath G., de la Motte R., Wagner F. W. 1989; Protease assay methods. Proteolytic Enzymes: a Practical Approach25–55 Beynon R. J., Bond S. J. Oxford: IRL Press at Oxford University Press;
    [Google Scholar]
  17. Scott J. H., Schekman R. 1980; Lyticase: endoglucanase and protease activities that act together in yeast cell lysis. Journal of Bacteriology 142:414–423
    [Google Scholar]
  18. Ugalde U., Pitt D. 1983; Morphology and calcium-induced conidiation of Penicillium cyclopium in submerged culture. Transactions of the British Mycological Society 80:319–325
    [Google Scholar]
  19. Ugalde U. O., Virto M. D., Pitt D. 1990; Calcium binding and induction of conidiation in protoplasts of Penicillium cyclopium . Antonie van Leeuwenhoek 57:43–49
    [Google Scholar]
  20. Vallee B. L., Ulmer D. D. 1972; Biochemical effects of mercury, cadmium and lead. Annual Review of Biochemistry 41:91–128
    [Google Scholar]
  21. Van der Valk H. C. P. M. 1984; Determination of proteases in isolated washed protoplasts: inactivation of proteases in cell wall-degrading enzyme mixtures used in protoplasts isolation. Plant Science Letters 36:201–204
    [Google Scholar]
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