1887

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Volume 53, Issue 1

The Protease Liberated from Strain H 18 by Mechanical Disintegration Free

Abstract

SUMMARY: Disintegration of lyophilized H 18 suspended in water, by agitation with glass beads, gave an extract containing 40% of the total cell protease activity. Better yields of an esterase which hydrolysed -toluenesulphonyl--arginine methyl ester (TAME) were obtained by disintegration in 0.1 M-phosphate buffer (pH 7.0). Ultrasonic disintegration of fresh suspensions was used to obtain larger quantities of cell extract. The protease had a broad plateau of activity between pH 5.5 and 9.5; the esterase had maximum activity at pH 8.0. Divalent cations had relatively little effect on either activity but 5 × 10 M-CaCl restored activity to EDTA-inhibited esterase. The protease was not inhibited by EDTA. Both activities were inhibited by di-isopropylphosphofluoridate but the protease was incompletely inhibited (87%). Neither activity was activated nor inhibited by thiol reagents. In addition to TAME, -α-benzoyl-L-arginine methyl ester (BAME), -α-benzoyl--arginine ethyl ester (BAEE), -α-benzoyl--arginine--nitroanilide and lysine ethyl ester were hydrolysed, indicating a trypsin-like specificity. The esterase differed from trypsin in not hydrolysing N-α-benzoyl--arginine amide (BAA) nor -α-benzoyl--arginine-naphthylamide (BANA) and in hydrolysing BAME more rapidly than TAME. There was some hydrolysis of -α-benzoyl--leucyl-2-naphthylamide and some amino peptidase activity as shown by the hydrolysis of L-analyl-2-naphthylamide, L-leucylglycine and L-leucinamide. TAME competitively inhibited at least 64% of the protease activity. The for casein was 0.17% (w/v). Casein at concentrations greater than 3.0% (w/v) caused substrate inhibition. The rate of ‘tyrosine’ liberation was proportional to protease concentration provided that less than 0.5 mg. ‘tyrosine’ was liberated from the 80 mg. casein in the standard assay. Protease concentration to the power 2/3 or the power 1/2 was proportional to the rate of hydrolysis but the straight line did not go to the origin.

H 18 extracts contained much nucleic acid which could not be separated from the protease activity by ammonium sulphate, protamine sulphate nor manganese chloride precipitation. Continuous electrophoresis was also ineffective. Ion-exchange chromatography separated the nucleic acids, but the protease activity was scattered in so many fractions that the purification was only three-fold and the recoveries poor.

  • Accepted:
  • Published Online:
© Society for General Microbiology 1968
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1968-08-01
2023-11-28
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References

  1. Anson M. L. 1938; The estimation of pepsin, trypsin, papain and cathepsin with haemoglobin.. J. gen. Physiol 22:79
     [Google Scholar]
  2. Blackburn T. H. 1968; Protease production by Bacteroides amylophilus strain h18.. J. gen. Microbiol 53:27
     [Google Scholar]
  3. Blackburn T. H., Hobson P. N. 1962; Further studies on the isolation of proteolytic bacteria from the sheep rumen.. J. gen. Microbiol 29:69
     [Google Scholar]
  4. Conway E. J. 1957 Microdiffusion Analysis and Volumetric Error 4th ed. London: Crosby Lockwood and Son Ltd.;
     [Google Scholar]
  5. Ellinger G. M., Boyne E. B. 1965; Amino acid composition of some fish products and casein.. Br. J. Nutr 19:587
     [Google Scholar]
  6. Erlanger B. F., Kokowsky N., Cohen W. 1961; The preparation and properties of two new chromogenic substrates of trypsin.. Arch. Biochem. Biophys 95:271
     [Google Scholar]
  7. Grassman W., Heyde W. 1929; Alkalimetrische Mikrobestimmung der Aminosäuren und Peptide.. Hoppe-Seyler’s Z. physiol. Chem 183:32
     [Google Scholar]
  8. Hall F. F., Kunkel H. O., Prescott J. M. 1966; Multiple proteolytic enzymes of Bacillus licheniformis.. Arch. Biochem. Biophys 114:145
     [Google Scholar]
  9. Hannig K. 1961; Synthetische Mischung aus gleichen Teilen von: Bromphenolblau, Bromkresol-grün, Kresolrot, Säurefuchsin, und Kresolpurpur.. Z. analyt. Chem 181:244
     [Google Scholar]
  10. Heppel L. 1955; Separation of proteins from nucleic acids.. Meth. Enzymol 1:137
     [Google Scholar]
  11. Hopsu V. K., Glenner G. G. 1963; Further observations on histochemical esterase and amidase activities with similarities to trypsin.. J. Histochem. Cytochem 11:520
     [Google Scholar]
  12. Jansen E. F., Balls A. K. 1952; The inhibition of β-and γ-chymotrypsin and trypsin by diiso-propylfluorophosphate.. J. biol. Chem 194:721
     [Google Scholar]
  13. Korkes S., del Campillo A., Gunsalus I. C., Ochoa S. 1951; Enzymatic synthesis of citric acid. IV. Pyruvate as acetyl donor.. J. biol. Chem 193:721
     [Google Scholar]
  14. Layne E. 1957; Spectrophotometric and turbidimetric methods for measuring proteins.. Meth. Enzymol 3:447
     [Google Scholar]
  15. Lowry O. H., Rosebrough N. J., Farr A. L., Randall R. J. 1951; Protein measurement with the Folin phenol reagent.. J. biol. Chem 193:265
     [Google Scholar]
  16. McDonald C. E., Chen L. L. 1965; The Lowry modification of the Folin reagent for determination of proteinase activity.. Analyt. Biochem 10:175
     [Google Scholar]
  17. McDonald I. J. 1962; Location of proteinase in cells of a species of micrococcus.. Can. J. Microbiol 8:785
     [Google Scholar]
  18. Miller B. S., Johnson J. A. 1951; A simple linear relationship and definition of a unit of proteinase activity.. Archs. Biochem. Biophys 32:200
     [Google Scholar]
  19. Mycek M. J., Elliott S. D., Fruton J. S. 1952; The specificity of a crystalline streptococcal proteinase.. J. biol. Chem 197:637
     [Google Scholar]
  20. Nathans D., Lipmann F. 1961; Amino acid transfer from aminoacyl-ribonucleic acids to protein on ribosomes of Escherichia coli.. Proc. natn. Acad. Sci. U.S.A 47:497
     [Google Scholar]
  21. Peterson E. A., Sober H. A. 1957; Variable gradient device for chromatography.. Analyt. Chem 31:857
     [Google Scholar]
  22. Peterson E. A., Sober H. A. 1962; Column chromatography of proteins: substituted celluloses.. Meth. Enzymol 5:3
     [Google Scholar]
  23. Sandvik O. 1962 Studies on Casein Precipitating Enzymes of Aerobic and Facultatively Anaerobic Bacteria Oslo: Veterinary College of Norway.;
     [Google Scholar]
  24. Schütz E. 1885; Eine Methode zur Bestimmung der relativen Pepsinmenge.. Hoppe-Seylers Z. physiol. Chem 9:577
     [Google Scholar]
  25. Schwert G. W., Neurath H., Kaufman S., Snoke J. E. 1948; The specific esterase activity of trypsin.. J. biol. Chem 172:221
     [Google Scholar]
  26. Smith E. L. 1955; Aminopeptidases.. Meth. Enzymol 2:83
     [Google Scholar]
  27. Weil-Malherbe H., Green R. H. 1955; Ammonia formation in brain. 1. Studies on slices and suspensions.. Biochem. J 61:210
     [Google Scholar]
  28. Werle E., Boetsch B. 1960; Über esteratische Wirkungen von Kallikrein und Trypsin und ihre Hemmung durch Kallikrein- und Tripsininhibitoren.. Hoppe-Seyler’s Z. physiol. Chem 319:52
     [Google Scholar]
  29. Zahn R. K., Stahl I. 1953; Die kontinuierliche Extraktion von Stoffgemischen unter Änderung eines Parameters nach dem Volum-Ersatzprinzip.. Hoppe-Seyler’s Z. physiol. Chem 293:1
     [Google Scholar]
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The Protease Liberated from Bacteroides amylophilus Strain H 18 by Mechanical Disintegration
Microbiology 53, 37 (1968); https://doi.org/10.1099/00221287-53-1-37
/content/journal/micro/10.1099/00221287-53-1-37
/content/journal/micro/10.1099/00221287-53-1-37
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