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Cold-active serine alkaline protease from the psychrophilic bacterium Pseudomonas strain DY-A: enzyme purification and characterization

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An extracellular protease was purified from a deep-sea psychrophilic bacterium strain DY-A which was identified as a Pseudomonas species. The optimal growth and protease-producing temperatures of the strain were all 10°C, and the protease was secreted only at temperatures under 20°C. The enzyme was most active at 40°C and at pH 10.0. It was inhibited by phenylmethyl sulfonylfluoride and diisopropyl fluorophosphate, indicating that it is a serine protease. Chelators such as EDTA, EGTA, 1,10-phenanthroline and 2,2′-bipyridyl produced a decrease of activity. The enzyme was sensitive to denaturing agents such as SDS, urea, and guanidine HCl and resistant to thiol-containing reducing agents such as dithiotreitol. The enzyme was active towards N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide. The native molecular mass of the enzyme determined by native PAGE and SDS-PAGE was 25 kDa.

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  • Chessa JP, Petrescu I, Bentahir M, Van Beeumen J, Gerday C (2000) Purification, physico-chemical characterization and sequence of a heat labile alkaline metalloprotease isolated from a psychrophilic Pseudomonas species. Biochim Biophys Acta 1479:265–274

    CAS  PubMed  Google Scholar 

  • Feller G, Narinx E, Arpigny JL, Aittaleb M, Baise E, Genicot S, Gerday C (1996) Enzymes from psychrophilic organisms. FEMS Microbiol Rev 18:189–202

    CAS  Google Scholar 

  • Gerday C, Aittaleb M, Arpigny JL, Baise E, Chessa JP, Garsoux G, Petrescu I, Feller G (1997) Psychrophilic enzymes: a thermodynamic challenge. Biochim Biophys Acta 1342:119–131

    CAS  PubMed  Google Scholar 

  • Hoshino T, Ishizaki K, Sakamoto T, Kumeta H, Yumoto I, Matsuyama H, Ohgiya S (1997) Isolation of a Pseudomonas species from fish intestine that produces a protease active at low temperature. Lett Appl Microbiol 25:70–72

    CAS  PubMed  Google Scholar 

  • Kulakova L, Galkin A, Kurihara T, Yoshimura T, Esaki N (1999) Cold-active serine alkaline protease from the psychrotrophic bacterium Shewanella strain Ac10: gene cloning and enzyme purification and characterization. Appl Environ Microbiol 65:611–617

    CAS  PubMed  Google Scholar 

  • Margesin R, Plama N, Knauseder F, Schinner F (1991) Proteases of psychrotrophic bacteria isolates from glaciers. J Basic Microbiol 31:377–383

    CAS  Google Scholar 

  • Vazquez SC, Rios Merino LN, MacCormack WP, Fraile ER (1995) Protease-producing psychrotrophic bacteria isolated from Antarctica. Polar Biol 15:131–135

    Google Scholar 

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This work was supported by NKBRSF Project G2000078500 and COMRA Project DY105-4-2-4.

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Correspondence to Runying Zeng.

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Communicated by K. Horikosui

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Zeng, R., Zhang, R., Zhao, J. et al. Cold-active serine alkaline protease from the psychrophilic bacterium Pseudomonas strain DY-A: enzyme purification and characterization. Extremophiles 7, 335–337 (2003).

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