Applied Microbiology and Biotechnology

, Volume 28, Issue 4–5, pp 409–413 | Cite as

Thermostable alkaline protease produced by Bacillus thermoruber — a new species of Bacillus

  • Pier L. Manachini
  • Maria G. Fortina
  • Carlo Parini


The proteolytic activity produced by a new species of Bacillus isolated in our laboratory was investigated. This enzyme was purified to homogeneity from cell-free culture liquids of B. thermoruber. The purification procedure included ion-exchange chromatography on DEAE-Sephadex A-50 and α-casein agarose affinity chromatography. The protease consists of one polypeptide chain with a molecular weight of 39000±800. the isoelectric point was 5.3; the optimum pH and temperature for proteolytic activity (on casein) was found to be pH 9 and 45°C respectively. Enzyme activity was inhibited by PMSF and EDTA. The stability was considerably increased by addition of Ca2+, and the protease exhibited a relatively high thermal stability. The alkaline protease shows a preference for leucine in the carboxylic side of the peptide bond of the substrate. The K m value for benzyloxycarbonyl-Ala-Ala-Leu-p-nitroanilide was 2.5 mM.


Thermal Stability Bacillus Leucine PMSF Proteolytic Activity 
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  1. Andrews P (1964) Estimation of the molecular weights of proteins by Sephadex gel filtration. Biochem J 91:222–232Google Scholar
  2. Cheetham PSJ (1985) The applications of enzymes in industry. In: Wiseman A (ed) Handbook of enzyme biotechnology. Ellis Horwood Limited Publ., Chichester, London, pp 274–379Google Scholar
  3. Debabov VG (1982) The industrial use of Bacilli. In: Dubnau DA (ed) The molecular biology of the Bacilli. Academic Press, New York, pp 331–370Google Scholar
  4. Gnosspelius G (1978) Purification and properties of an extracellular protease from Myxococcus virescens. J Bacteriol 133:17–25Google Scholar
  5. Izotova LS, Strongin AY, Chekulaeva LN, Sterkin VE, Ostoslavskaya VI, Lyublinskaya LA, Timokhina EA, Stepanov VM (1983) Purification and properties of serine protease from Halobacterium halobium. J Bacteriol 155:826–830Google Scholar
  6. Kato N, Adachi S, Takeuchi K, Morihara K, Tani Y, Ogata K (1974) Substrate specificities of the protease from a marine-psychrophilic bacterium, Pseudomonas sp No 548. Agric Biol Chem 38:103–109Google Scholar
  7. Kelly CT, Fogarty WM (1976) Microbial alkaline enzymes. Process Biochemistry 11:3–9Google Scholar
  8. Laemmli UK (1970) Cleavage of structure proteins during assembly of the head of bacteriophage T4. Nature 277:680–685Google Scholar
  9. Lowry OH, Rosebrough NJ, Farr AF, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275Google Scholar
  10. Manachini PL, Fortina MG, Parini C, Craveri R (1985) Bacillus thermoruber sp. nov., nom. rev., a red-pigmented thermophilic bacterium. Int J Syst Bacteriol 35:493–496Google Scholar
  11. Moore WEC, Hash DE, Holdeman LV, Cato EP (1980) Polyacrylamide slab gel electrophoresis of soluble proteins for studies of bacterial floras. Appl Environ Microbiol 39:900–907Google Scholar
  12. Morihara K (1974) Comparative specificity of microbial proteinases. Adv Enzymol 41:179–243Google Scholar
  13. Strongin AYA, Izotova LS, Abramov ZT, Gorodetsky DI, Ermakova LM, Baratova LA, Belyanova LP, Stepanov VM (1978) Intracellular serine protease of Bacillus subtilis: sequence homology with extracellular subtilisins. J Bacteriol 133:1401–1411Google Scholar
  14. Strongin AYA, Abramov ZT, Yaroslavtseva NG, Baratova LA, Shaginyan KA, Belyanova LP, Stepanov VM (1979) Direct comparison of subtilisin-like intracellular protease of Bacillus licheniformis with the homologous enzymes of Bacillus subtilis. J Bacteriol 137:1017–1019Google Scholar
  15. Ward OP (1983) Proteinases. In: Fogarty WM (ed) Microbial enzymes and biotechnology. Applied Science Publ, New York, pp 251–317Google Scholar

Copyright information

© Springer-Verlag 1988

Authors and Affiliations

  • Pier L. Manachini
    • 1
  • Maria G. Fortina
    • 1
  • Carlo Parini
    • 1
  1. 1.Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, Sezione di Micrbiologia IndustrialeUniversità degli Studi di MilanoMilanoItalia

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