Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Purification and Characterization of Two Alkaline Proteases Produced by Pseudomonas sp. BK7

  • Published : 2000.10.01

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Abstract

Pseudomonas sp. BK7, an alkalophile, displayed the highest growth and protease activity when grown in a fermenter which was controlled at a pH level of 9.0, and the enzyme production was significantly enganced by the increase of agitation speed. Two formas of alkaline proteases (BK7-1 and BK7-2) were fractionated and purified to near homogeneity. Protease BK7-1 was purified through CM-Sepharose CL-6B and Sephadex G-75 column chromatographies, and Protease BK7-2 was purified through CM-Sepharose CL-6B and Sephadex G-75 column chromatographies, and Protease BK7-2 was purified through CM-Sepharose CL-6B, DEAE-Sepharose, and Sephadex G-75 column chromatographies. The molecular weights of proteases BK7-1 and BK7-2 determined by gel filtration chromatography were 20,700 and 40,800, respectively. The $K_m$ value, isoelectric point, and optimum pH of protease BK7-1 were 2.55 mg/ml, 11.0 and 11.0, respectively, whereas those of protease BK7-2 were 1.57 mg/ml, 7.2, and 10.0, respectively. Both protease were practically stable in the pH range of 5-11. The optimum temperatures for the activities of both protease BK7-1 and BK7-2 were 50℃ and 45℃, respectively. About 56% of the original protease BK7-2 activity remained after being treated at 50℃ for 30 min but protease BK7-1 was rapidly inactivated at above 25℃. Both proteases were completely inhibited by phenylmethane sulfonyl fluoride, a serine protease inhibitor. Protease BK7-2 was stable against EDTA, EGTA, STP, and detergents such as SDS and LAS, whereas protease BK7-1 was found to be unstable.

Keywords

References

  1. J. Bacteriol v.171 Isolation, Purification and properties of Penicillium charlesii alkaline protease Abbs, C. A.;S. Grones;J. E. Gander
  2. J. Biochem v.91 Estimation of molecular weight of protein by Sephadex gel filtration Andrews, T.
  3. Anal. Biochem v.72 A Rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein-dye binding Bradford, M.
  4. Can. J. Microbiol v.42 Purification and partial characterization of two extracelluar alkaline protease from Streptomyces corchorusii ST36 El-Shanshoury, A.;M. A. El-Sayed;W. A. El-Shouny
  5. J. Biotech v.30 Purification and properties of the highly thermostable alkaline protease from and alkaliphilic and the thermophilic Bacillus Fujiwara, N.;A. Masui
  6. The Application of Enzyme Industry: Industrial Enzymology Godfrey, T.;J. Reichelt
  7. J. Microbiol. Biotechnol v.9 Minor thermostable alkaline protease produced by Thermoactinomyces sp. E79 Kim, Y.-O.;J.-K. Lee;K. Sunita;H.-K. Kim;T.-K. Oh
  8. Appl. Microbiol. Biotechnol v.43 Purification and properties of an alkaline protease from alkalophilic Bacillus sp. KSM-K16 Kobayashi, T;Y. Hakamada;S. Sachi;J. hitomi;S. Ito
  9. Biotech. Letters v.16 Extracelluar alkaline proteases from alkalophilic Vibrio metschinikovii strain RH530 Kwon, Y. T.;J. O. Kim;S. Y. Moon;H. H. Lee;H. Y. Rho
  10. Kor. J. Microbiol v.22 Isolation and characterization of Pseudomonas sp. producing alkaline protease Lee, E. G.;E. H. Park;H. H. Lee;H. H. Hyun
  11. J. Biol. Chem v.256 Alkaline protease from Neurospora crassa. Purification and partial characterization Linberg, R. A.;L. D. Erich;J. S. Price;H. Durcker
  12. Appl. Microbiol. Biotechnol v.28 Thermostable alkaline protease produced by Bacillus thermoruber - a new species of Bacillus Manachini, P. L.;M. G. Fortina;C. Parini
  13. J. Biotech v.24 Purification and characterization of an alkaline serine protease produced by psychrotrophic Bacillus sp. Margesin, R.;N. Plama;F. Knauseder;F. Schinner
  14. App. Microbiol. Biotechnol v.40 Purification and characterization of a heat stable alkaline protease from Bacillus stearothermophilus F1 Rahman, R.;C. N. Rajak;K. Ampon;M. Basri;W. Yunus;A. B. Salleh
  15. J. Ferm. Bioeng v.77 Pusification and characterization of an alkaline protease from Oerskovia xanthineolytica TK-1 Saeki, K.;J. Iwata;Y. Watanabe;Y. Tamai
  16. Appl. Microbiol. Biotechnol v.48 Production, purification and characterization of a 50-kDa extracelluar metalloprotease from Serratia marcescens Salamon, P. R.;R. J. Wodzinski
  17. Agric. Biol. Chem v.55 Purification and properties of a novel surface-active agent-and alkaline- resistant protease from Bacillus sp.Y Shimogaki, H.;K. Takeuchi;M. Irie
  18. Ann. NY Acad. Sci. v.121 Spectrophotometric analyzers for disc electrophoresis: Studies of yeast cytochromec Taber, H. W.;F. Sherman
  19. Appl. Microbiol. Biotechnol v.30 Production of extremely thermostable alkaline protease from Bacillus sp.no. AH-101 Takami, H.;T. Akiba;K. Horikoshi
  20. Microbiol Enzymes and Biotechnology Ward, O. P.
  21. J. Microbiol. Biotechnol v.9 Akaline protease of a genetically-engineered Aspergillus oryzae for the use as a silver recovery agent from used X-ray film Warin, S.;M. Tanticharoen