Characterization of proteolytic bacteria from the Aleutian deep-sea and their proteases

  • Hairong Xiong
  • Linsheng Song
  • Ying Xu
  • Man-Yee Tsoi
  • Sergey Dobretsov
  • Pei-Yuan Qian
Original Paper

Abstract

Six deep-sea proteolytic bacteria taken from Aleutian margin sediments were screened; one of them produced a cold-adapted neutral halophilic protease. These bacteria belong to Pseudoalteromonas spp., which were identified by the 16S rDNA sequence. Of the six proteases produced, two were neutral cold-adapted proteases that showed their optimal activity at pH 7–8 and at temperature close to 35°C, and the other four were alkaline proteases that showed their optimal activity at pH 9 and at temperature of 40–45°C. The neutral cold-adapted protease E1 showed its optimal activity at a sodium chloride concentration of 2 M, whereas the activity of the other five proteases decreased at elevated sodium chloride concentrations. Protease E1 was purified to electrophoretic homogeneity and its molecular mass was 34 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of protease E1 was determined to be 32,411 Da by mass spectrometric analysis. Phenylmethyl sulfonylfluoride (PMSF) did not inhibit the activity of this protease, whereas it was partially inhibited by ethylenediaminetetra-acetic acid sodium salt (EDTA-Na). De novo amino acid sequencing proved protease E1 to be a novel protein.

Keywords

Deep-sea bacteria Halophilic Protease Pseudoalteromonas 

References

  1. 1.
    Arahal DR, Dewhirst FE, Paster BJ, Volcani BE, Ventosa A (1996) Phylogenetic analyses of some extremely halophilic archaea isolated from Dead Sea water, determined on the basis of their 16S rRNA sequences. Appl Environ Microbiol 62:3779–3786Google Scholar
  2. 2.
    Chen XL, Zhang YZ, Gao PJ, Luan XW (2003) Two different proteases produced by a deep-sea psychrotrophic bacterial, Pseudoaltermonas sp. SM9913. Mar Biol 143:989–993CrossRefGoogle Scholar
  3. 3.
    Ghorbel B, Sellami-Kamoun A, Nasri M (2003) Stability studies of protease from Bacillus cereus BG1. Enzyme Microb Technol 32:513–518CrossRefGoogle Scholar
  4. 4.
    Giménez MI, Studdert CA, Sánchez JJ, Castro RED (2000) Extracellular protease of Natrialba magadii: purification and biochemical characterization. Extremophiles 4:181–188CrossRefGoogle Scholar
  5. 5.
    Gupta R, Beg QK, Khan S, Chauhan B (2002) An overview on fermentation, downstream processing and properties of microbial alkaline proteases. Appl Microbiol Biotechnol 60:381–395CrossRefGoogle Scholar
  6. 6.
    Hiraga K, Nishikata Y, Namwong S, Tanasupawat S, Takada K, Oda K (2005) Purification and characterization of serine proteinase from a halophilic bacterium, Filobacillus sp. RF2–5. Biosci Biotechnol Biochem 69:38–44CrossRefGoogle Scholar
  7. 7.
    Huston AL, Krieger-Brockett BB, Demin JW (2000) Remarkably low temperature optima for extracellular enzyme activity from Arctic bacteria and sea ice. Environ Microbiol 2:383–388CrossRefGoogle Scholar
  8. 8.
    Ivanova EP, Bakunina Y, Nedashkovskaya OI, Gorshkova NM, Alexeeva YV, Zelepuga EA, Zvaygintseva TN, Nicolau DV, Mikhailov VV (2003) Ecophysiological variabilities in ectohydrolytic enzyme activities of some Pseudoalteromonas species, P. citrea, P. issachenkonii, and P. nigrifaciens. Curr Microbiol 46:6–10CrossRefGoogle Scholar
  9. 9.
    Kaye JZ, Baross JA (2004) Synchronous effects of temperature, hydrostatic pressure, and salinity on growth, phospholipid profiles, and protein patterns of four Halomonas species isolated from deep-sea hydrothermal-vent and sea surface environments. Appl Environ Microbiol 70:6220–6229CrossRefGoogle Scholar
  10. 10.
    Klibanov AM (2001) Improving enzymes by using them in organic solvents. Nature 409:241–246CrossRefGoogle Scholar
  11. 11.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bateriophage T4. Nature 227:680–685CrossRefGoogle Scholar
  12. 12.
    Lee SO, Kato J, Nakashima K, Kuroda A, Ikeda T, Takiguchi N, Ohtake H (2002) Cloning and characterization of extracellular metal protease gene of the algicidal marine bacterium Pseudoalteromonas sp. strain A28. Biosci Biotechnol Biochem 66:1366–1369CrossRefGoogle Scholar
  13. 13.
    Marhuenda-Egea FC, Bonete MJ (2002) Extreme halophilic enzymes in organic solvents. Curr Opin Biotechnol 13:385–389CrossRefGoogle Scholar
  14. 14.
    Miyamoto K, Tsujibo H, Nukui E, Itoh H, Kaidzu Y, Inamori Y (2002) Isolation and characterization of the genes encoding two metalloproteases (MprI and MprII) from a marine bacterium, Alteromonas sp. strain O-7. Biosci Biotechnol Biochem 66:416–421CrossRefGoogle Scholar
  15. 15.
    O’Brien A, Sharp R, Russell NJ, Roller S (2004) Antarctic bacteria inhibit growth of food-borne microorganisms at low temperatures. FEMS Microbiol Ecol 48:157–167CrossRefGoogle Scholar
  16. 16.
    Rao MB, Tanksale AM, Ghatge MS, Deshpande VV (1998) Molecular and biotechnological aspects of microbial proteases. Microbiol Mol Biol Rev 62:597–635Google Scholar
  17. 17.
    Ryu K, Kim J, Dordick JS (1994) Catalytic properties and potential of an extracellular protease from an extreme halophile. Enzyme Microb Technol 16:266–275CrossRefGoogle Scholar
  18. 18.
    Su NW, Lee MH (2001) Purification and characterization of a novel salt-tolerant protease from Aspergillus sp. FC-10, a soy sauce koli mold. J Ind Microbiol Biotechnol 26:254–258Google Scholar
  19. 19.
    Sánchez-Porro C, Mellado E, Bertoldo C, Antranikian G, Ventosa A (2003) Screening and characterization of the protease CP1 produced by the moderately halophilic bacterium Pseudoalteromonas sp. StrainCP76 Extremophiles 7:221–228Google Scholar
  20. 20.
    Turunen O, Jänis J, Fenel F, Leisola M (2004) Engineering the thermotolerance and pH optimum of family 11 xylanases by site-directed mutagenesis. Methods Enzymol 388:156–167CrossRefGoogle Scholar
  21. 21.
    Wang SL, Chen YH, Wang CL, Yen YH, Chern MK (2005) Purification and characterization of a serine protease extracellularly produced by Aspergillus fumigatus in a shrimp and crab shell powder medium. Enzyme Microb Technol 36:660–665CrossRefGoogle Scholar
  22. 22.
    Xiong H, Nyyssölä A, Jänis J, Pastinen O, Weymarn N, Leisola M, Turunen O (2004a) Characterization of the xylanase produced on submerged cultivation by Thermomyces lanuginosus DSM 10635. Enzyme Microb Technol 35:93–99CrossRefGoogle Scholar
  23. 23.
    Xiong H, Fenel F, Leisola M, Turunen O (2004b) Engineering the thermostability of Trichoderma reesei endo-β-1,4 xylanase II by combination of disulphide bridges. Extremophiles 8:393–400 CrossRefGoogle Scholar
  24. 24.
    Yebra DM, Kiil S, Dam-Johansen K (2004) Antifouling technology—past, present and future steps towards efficient and environmentally friendly antifouling coatings. Prog Org Coat 50:75–104CrossRefGoogle Scholar
  25. 25.
    Zeng R, Zhang R, Zhao J, Lin N (2003) Cold-active serine alkaline protease from the psychrophilic bacterium Pseudomonas strain DY-A: enzyme purification and characterization. Extremophiles 7:335–337CrossRefGoogle Scholar

Copyright information

© Society for Industrial Microbiology 2006

Authors and Affiliations

  • Hairong Xiong
    • 1
  • Linsheng Song
    • 2
  • Ying Xu
    • 1
  • Man-Yee Tsoi
    • 1
  • Sergey Dobretsov
    • 1
  • Pei-Yuan Qian
    • 1
  1. 1.Coastal Marine Laboratory, Department of BiologyHong Kong University of Science and TechnologyHong Kong SARChina
  2. 2.Experimental Marine Biology Laboratory, Institute of OceanologyChinese Academy of SciencesQindaoChina

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