Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

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

  • 139 Accesses

  • 97 Citations


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.

This is a preview of subscription content, log in to check access.


  1. Andrews P (1964) Estimation of the molecular weights of proteins by Sephadex gel filtration. Biochem J 91:222–232

  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 274–379

  3. Debabov VG (1982) The industrial use of Bacilli. In: Dubnau DA (ed) The molecular biology of the Bacilli. Academic Press, New York, pp 331–370

  4. Gnosspelius G (1978) Purification and properties of an extracellular protease from Myxococcus virescens. J Bacteriol 133:17–25

  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 155:826–830

  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 38:103–109

  7. Kelly CT, Fogarty WM (1976) Microbial alkaline enzymes. Process Biochemistry 11:3–9

  8. Laemmli UK (1970) Cleavage of structure proteins during assembly of the head of bacteriophage T4. Nature 277:680–685

  9. Lowry OH, Rosebrough NJ, Farr AF, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275

  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 35:493–496

  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 39:900–907

  12. Morihara K (1974) Comparative specificity of microbial proteinases. Adv Enzymol 41:179–243

  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 133:1401–1411

  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 137:1017–1019

  15. Ward OP (1983) Proteinases. In: Fogarty WM (ed) Microbial enzymes and biotechnology. Applied Science Publ, New York, pp 251–317

Download references

Author information

Correspondence to Pier L. Manachini.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Manachini, P.L., Fortina, M.G. & Parini, C. Thermostable alkaline protease produced by Bacillus thermoruber — a new species of Bacillus . Appl Microbiol Biotechnol 28, 409–413 (1988). https://doi.org/10.1007/BF00268205

Download citation


  • Thermal Stability
  • Bacillus
  • Leucine
  • PMSF
  • Proteolytic Activity