, Volume 8, Issue 6, pp 489–498 | Cite as

Thermoactive extracellular proteases of Geobacillus caldoproteolyticus, sp. nov., from sewage sludge

  • Xiao-Ge Chen
  • Olena Stabnikova
  • Joo-Hwa Tay
  • Jing-Yuan Wang
  • Stephen Tiong-Lee Tay
Original Paper


A proteolytic thermophilic bacterial strain, designated as strain SF03, was isolated from sewage sludge in Singapore. Strain SF03 is a strictly aerobic, Gram stain-positive, catalase-positive, oxidase-positive, and endospore-forming rod. It grows at temperatures ranging from 35 to 65°C, pH ranging from 6.0 to 9.0, and salinities ranging from 0 to 2.5%. Phylogenetic analyses revealed that strain SF03 was most similar to Saccharococcus thermophilus, Geobacillus caldoxylosilyticus, and G. thermoglucosidasius, with 16S rRNA gene sequence identities of 97.6, 97.5 and 97.2%, respectively. Based on taxonomic and 16S rRNA analyses, strain SF03 was named G. caldoproteolyticus sp. nov. Production of extracellular protease from strain SF03 was observed on a basal peptone medium supplemented with different carbon and nitrogen sources. Protease production was repressed by glucose, lactose, and casamino acids but was enhanced by sucrose and NH4Cl. The cell growth and protease production were significantly improved when strain SF03 was cultivated on a 10% skim-milk culture medium, suggesting that the presence of protein induced the synthesis of protease. The protease produced by strain SF03 remained active over a pH range of 6.0–11.0 and a temperature range of 40–90°C, with an optimal pH of 8.0–9.0 and an optimal temperature of 70–80°C, respectively. The protease was stable over the temperature range of 40–70°C and retained 57 and 38% of its activity at 80 and 90°C, respectively, after 1 h.


Characterization Extracellular Geobacillus caldoproteolyticus Thermoactive protease Thermophilic 


  1. Ahmad S, Scopes RK, Rees GN, Patel BKC (2000) Saccharococcus caldoxylosilyticus sp. nov., an obligately thermophilic, xylose-utilizing, endospore-forming bacterium. Int J Syst Evol Microbiol 50:517–523PubMedGoogle Scholar
  2. Antranikian G, Klingeberg M (1991a) Thermostable protease from Thermococcus. PCT Patent Appl WO 9119792Google Scholar
  3. Antranikian G, Klingeberg M (1991b) Thermostable protease from Staphylothermus. PCT Patent Appl WO 9119791Google Scholar
  4. Anwar A, Saleemuddin M (1998) Alkaline proteases: a review. Bioresource Technol 64:175–183CrossRefGoogle Scholar
  5. Ash C, Farrow JAE, Wallbanks S, Collins MD (1991) Phylogenetic heterogeneity of the genus Bacillus revealed by comparative analysis of small-subunit-ribosomal RNA sequences. Lett Appl Microbiol 13:202–206Google Scholar
  6. Boethling RS (1975) Regulation of extracellular protease secretion in Pseudomonas maltophilia. J Bacteriol 123:954–961PubMedGoogle Scholar
  7. Cowan DA, Smolenski KA, Daniel RM, Morgan HW (1987) An extremely thermostable extracellular proteinase from a strain of the archaebacterium Desulfurococcus growing at 88°C. Biochem J 247:121–133PubMedGoogle Scholar
  8. Denkin SM, Nelson DR (1999) Induction of protease activity in Vibrio anguillarum by gastrointestinal mucus. Appl Environ Microbiol 65:3555–3560Google Scholar
  9. Dhandapani R, Vijayaragavan R (1994) Production of a thermophilic extracellular alkaline protease by Bacillus stearothermophilus AP-4. World J Microbiol Biotechnol 10:33–35Google Scholar
  10. Eggen R, Geerling A, Watts J, de Vos WM (1990) Characterization of pyrolysin, a hyperthermoactive serine protease from the archaeobacterium Pyrococcus furiosus. FEMS Microbiol Lett 71:17–20CrossRefGoogle Scholar
  11. Felsenstein J (1989) PHYLIP (PHYlogenetic Inference Package). Cladistics 5:164–166Google Scholar
  12. Ferrero MA, Castro GR, Abate CM, Baigorí MD, Siñeriz F (1996) Thermostable alkaline proteases of Bacillus licheniformis MIR 29: isolation, production and characterization. Appl Microbiol Biotechnol 45:327–332Google Scholar
  13. Fortina MG, Mora D, Schumann P, Parini C, Manachini PL, Stackebrandt E (2001) Reclassification of Saccharococcus caldoxylosilyticus as Geobacillus caldoxylosilyticus (Ahmad et al. 2000) comb. nov. Int J Syst Evol Microbiol 51:2063–2071PubMedGoogle Scholar
  14. Godfrey T and West S (1996) Introduction to industrial enzymology. In: Godfrey T, West S (eds) Industrial enzymology, 2nd edn. Macmillan, London, pp 1–8Google Scholar
  15. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98Google Scholar
  16. Heinen UJ, Heinen W (1972) Characteristics and properties of a caldoactive bacterium producing extracellular enzymes and two related strains. Arch Mikrobiol 82:1–23PubMedGoogle Scholar
  17. Kalisz HM (1988) Microbial proteinases. Adv Biochem Eng Biotechnol 36:1–65PubMedGoogle Scholar
  18. Kumar CG, Takagi H (1999) Microbial alkaline proteases: from a bioindustrial viewpoint. Biotechnol Adv 17:561–594CrossRefPubMedGoogle Scholar
  19. Lane DJ (1991) 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M (eds) Nucleic acid techniques in bacterial systematics. Wiley, Chichester, pp 115–175Google Scholar
  20. Lee JK, Kim YO, Kim HK, Park YS, Oh TK (1996) Purification and characterization of a thermostable alkaline protease from Thermoactinomyces sp. E79 and the DNA sequence of the encoding gene. Biosci Biotechnol Biochem 60:840–846Google Scholar
  21. Maidak BL, Cole JR, Lilburn TG, Parker CT Jr, Saxman PR, Stredwick JM, Garrity GM, Li B, Olsen GJ, Pramanik S, Schmidt TM, Tiedje JM (2000) The RDP (Ribosomal Database Project) continues. Nucleic Acids Res 28:173–174CrossRefPubMedGoogle Scholar
  22. Manachini PL, Mora D, Nicastro G, Parini C, Stackebrandt E, Pukall R, Fortina MG (2000). Bacillus thermodenitrificans sp. nov., nom. rev. Int J Syst Evol Microbiol 50:1331–1337PubMedGoogle Scholar
  23. Matsuzawa H, Hamaoki M, Ohta T (1983) Production of thermophilic extracellular proteases (aqualysins I and II) by Thermus aquaticus YT-1. Agric Biol Chem 47:25–28Google Scholar
  24. Morikawa M, Izawa Y, Rashid N, Hoaki T, Imanaka T (1994) Purification and characterization of a thermostable thiol protease from a newly isolated hyperthermophilic Pyrococcus sp. Appl Environ Microbiol 60:4559–4566Google Scholar
  25. Nazina TN, Tourova TP, Poltaraus AB, Novikova EV, Grigoryan AA, Ivanova AE, Lysenko AM, Petrunyaka VV, Osipov GA, Belyaev SS, Ivanov MV (2001) Taxonomic study of aerobic thermophilic bacilli: descriptions of Geobacillus subterraneus gen. nov., sp. nov. and Geobacillus uzenensis sp. nov. from petroleum reservoirs and transfer of Bacillus stearothermophilus, Bacillus thermocatenulatus, Bacillus thermoleovorans, Bacillus kaustophilus, Bacillus thermoglucosidasius and Bacillus thermodenitrificans to Geobacillus as the new combinations G. stearothermophilus, G. thermocatenulatus, G. thermoleovorans, G. kaustophilus, G. thermoglucosidasius and G. thermodenitrificans. Int J Syst Evol Microbiol 51:433–446PubMedGoogle Scholar
  26. Nystrand R (1984) Saccharococcus thermophilus gen. nov., sp. nov., isolated from beet sugar extraction. Syst Appl Microbiol 5:204–219Google Scholar
  27. Peek K, Daniel RM, Monk C, Parker L, Coolbear T (1992) Purification and characterization of a thermostable proteinase isolated from Thermus sp. strain Rt41A. Eur J Biochem 207:1035–1044PubMedGoogle Scholar
  28. Rahman RNZA, Razak CN, Ampon K, Basri M, Yunus WMZW, Salleh AB (1994) Purification and characterization of a heat-stable alkaline protease from Bacillus stearothermophilus F1. Appl Microbiol Biotechnol 40:822–827Google Scholar
  29. Rainey FA, Stackebrandt E (1993) Phylogenetic evidence for the relationship of Saccharococcus thermophilus to Bacillus stearothermophilus. Syst Appl Microbiol 16:224–226Google Scholar
  30. Rainey FA, Fritze D, Stackebrandt E (1994). The phylogenetic diversity of thermophilic members of the genus Bacillus as revealed by 16S rDNA analysis. FEMS Microbiol Lett 115:205–211CrossRefPubMedGoogle Scholar
  31. Secades P, Guijarro JA (1999) Purification and characterization of an extracellular protease from the fish pathogen Yersinia ruckeri and effect of culture conditions on production. Appl Environ Microbiol 65: 3969–3975Google Scholar
  32. Smiber RM, Krieg NR (1994) Phenotypic characterization. In: Gerhardt P, Murray RGE, Wood WA, Krieg NR (eds) Methods for general and molecular bacteriology. American Society for Microbiology, Washington, pp 607–654Google Scholar
  33. Sneath PHA (1986) Endospore-forming Gram-positive rods and cocci. In: Sneath PHA, Mair NS, Sharp ME, Holt JG (eds) Bergey’s manual of systematic bacteriology, vol 2. Williams and Wilkins, Baltimore, pp 1104–1139Google Scholar
  34. Stackebrandt E, Goebel BM (1994) Taxonomic note: a place for DNA–DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44:846–849Google Scholar
  35. Sunna A, Tokajian S, Burghardt J, Rainey F, Antranikian G, Hashwa F (1997) Identification of Bacillus kaustophilus, Bacillus thermocatenulatus and Bacillus strain HSP as members of Bacillus thermoleovorans. Syst Appl Microbiol 20:232–237Google Scholar
  36. Suzuki Y, Kishigami T, Inoue K, Mizoguchi Y, Eto N, Takagi M, Abe S (1983) Bacillus thermoglucosidasius sp. nov., a new species of obligately thermophilic bacilli. Syst Appl Microbiol 4:487–495Google Scholar
  37. Tsuchiya K, Nakamura Y, Sakashita H, Kimura T (1992) Purification and characterization of thermostable alkaline protease from alkalophilic Thermoactinomyces sp HS682. Biosci Biotechnol Biochem 56:246–250Google Scholar
  38. Ursing JB, Rosselló-Mora RA, García-Valdés E, Lalucat J (1995) Taxonomic note: a pragmatic approach to the nomenclature of phenotypically similar genomic groups. Int J Syst Bacteriol 45:604Google Scholar
  39. Voordouw G, Roche RS (1975) The role of bound calcium ions in thermostable, proteolytic enzymes. II. Studies on thermolysin, the thermostable protease from Bacillus thermoproteolyticus. Biochemistry 14:4667–4673Google Scholar
  40. Yoshikawa M, Matsuda F, Naka M, Murofushi E, Tsunematsu. J (1974) Pleiotropic alterations of activities of several toxins and enzymes in mutants of Staphylococcus aureus. J Bacteriol 119:117–122PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • Xiao-Ge Chen
    • 1
    • 2
  • Olena Stabnikova
    • 1
  • Joo-Hwa Tay
    • 1
  • Jing-Yuan Wang
    • 1
  • Stephen Tiong-Lee Tay
    • 1
  1. 1.Environmental Engineering Research Center, School of Civil and Environmental EngineeringNanyang Technological UniversitySingapore
  2. 2.Institute of Environmental Science and EngineeringNanyang Technological UniversitySingapore

Personalised recommendations