World Journal of Microbiology and Biotechnology

, Volume 24, Issue 2, pp 237–243

Biosynthesis and properties of an extracellular thermostable serine alkaline protease from Virgibacillus pantothenticus

Original Paper


In this communication, we report the presence of a newly identified serine alkaline protease producing bacteria, Virgibacillus pantothenticus (MTCC 6729) in the fresh chicken meat samples and the factors affecting biosynthesis as well as characterization of protease. The strain produced only 14.3 U ml−1 protease in the standard medium after 72 h of incubation, while in optimized culture conditions the production of protease was increased up to 18.2 U ml−1. The strain was able to produce protease at 40°C at pH 9.0. The addition of dextrose and casein improved protease production. The protease was partially purified and characterized in terms of pH and temperature stability, effect of metal ions and inhibitors. The protease was found to be thermostable alkaline by retaining its 100% and 85% stability at pH 10.0 and at 50°C respectively. The protease was compatible with some of the commercial detergents tested, and was effective in removing protein stains from cotton fabrics. The V. pantothenticus, MTCC 6729 protease appears to be potentially useful as an additive in detergents as a stain remover and other bio-formulations.


Alkaline protease Detergent compatibility Enzyme Serine protease Virgibacilluspantothenticus 


  1. Anwar A, Saleemuddin M (1997) Alkaline pH acting digestive enzymes of the polyphagous insect pest Spilosoma obliqua: stability and potential as detergent additives. Biotechnol Appl Biochem 25:43–46Google Scholar
  2. Borris R (1987) Biology of enzymes. In: Rehm H, Reed G (eds) Biotechnology. Weinheim, Verlag chemie, pp 35–62Google Scholar
  3. Cowan D (1996) Industrial enzyme technology. Trends Biotechnol 14:177–178CrossRefGoogle Scholar
  4. Damare S, Raghukumar C, Muraleedharan UD, Raghukumar S (2006) Deep-sea fungi as a source of alkaline and cold-tolerant proteases. Enzyme Microb Technol 39:172–181CrossRefGoogle Scholar
  5. El-Hawary FI, Ibrahim II (1992) Comparative study on protease of three thermophilic Bacilli. Zagazig J Agric Res 19:777–787Google Scholar
  6. Feng YY, Yang WB, Ong SL, Hu JY, Ng WJ (2001) Fermentation of starch for enhanced alkaline protease production by constructing an alkalophilic Bacillus pumilus strain. Appl Microbiol Biotechnol 57:153–160CrossRefGoogle Scholar
  7. Gaure R, Yadav J, Pandey L (1989) Thermostbility of extracellular protease enzyme produced by Spicaria fusispora. Hindustan Antibiot Bull 31:36–37Google Scholar
  8. Gold AM, Fahrney D (1964) Sulfonyl fluorides as inhibitors of esterases. II. Formation and reactions of phenylmethane sulfonyl alpha-chymotrypsin. Biochemistry 3:783–791 CrossRefGoogle Scholar
  9. Gupta R, Beg Q, Khan S, Chauhan B (2002) An overview on fermentation, downstream processing and properties of microbial alkaline protease. Appl Microbiol Biotechnol 60:381–395CrossRefGoogle Scholar
  10. Heyndrickx M, Lebbe L, Kersters K, De Vos P, Forsyth G, Logan NA (1998) Virgibacillus: a new genus to accommodate Bacillus pantothenticus (Proom and Knight 1950). Emended description of Virgibacillus pantothenticus. Int J Syst Bacteriol 48:99–106CrossRefGoogle Scholar
  11. Holt JG, Krieg NR, Sneath PHA, Stately JT, Williams ST (1994) Bergey’s manual of determinative bacteriology, 9th edn. Williams and Wilkins, Baltimore, pp 787Google Scholar
  12. Jang JS, Kang DO, Chun MJ, Byun SM (1992) Molecular cloning of a subtilisin J gene from Bacillus stearothermophilus and its expression in Bacillus subtilis. Biochem Biophys Res Commun 184:277–282CrossRefGoogle Scholar
  13. Kanekar P, Nilegaonkar S, Sarnaik S, Kelkar A (2002) Optimization of protease activity of alkalisphilic bacteria isolated from an alkaline lake in India. Bioresour Technol 85:87–93CrossRefGoogle Scholar
  14. Kaneko R, Koyama N, Tsai Y, Juang R, Yoda K, Yamasaki M (1989) Molecular cloning of the structural gene for alkaline elastase YaB, a new subtilisin produced by an alkalophilic Bacillus strain. J Bacteriol 171:5232–5236Google Scholar
  15. Kim J, Lim W, Suh H (2001) Feather-degrading Bacillus species from poultry waste. Process Biochem 37:287–291CrossRefGoogle Scholar
  16. Liang T, Lin J, Yen Y, Wang C, Wang S (2006) Purification and characterization of a protease extracellularly produced by Monascus purpureus CCRC31499 in a shrimp and crab shell powder medium. Enzyme Microb Technol 38:74–80CrossRefGoogle Scholar
  17. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265–275Google Scholar
  18. MacFarlane G, MacFarlane S, Gibson G (1992) Synthesis and release of protease by Bacteroide fragilis. Curr Microbiol 24:55–59CrossRefGoogle Scholar
  19. Mala B, Rao Aparna M, Deshpade VV (1998) Molecular and biotechnological aspects of microbial proteases . Microbiol Mol Biol Rev 62:597–635Google Scholar
  20. Manachini PL, Fortina MG, Parini C (1988) Alkaline protease produced by Bacillus thermoruber a new species of Bacillus. Appl Microbiol Biotechnol 28:409–413CrossRefGoogle Scholar
  21. Nilegaonkar S, Kanekar P, Sarnaik S, Kelkay A (2002) Production, isolation and characterization of extracellular protease of analkaliphilic strain of Arthrobacter ramosus, MCM B-351 isolated from the alkaline lake of lonar, India. World J Microbol Biotechnol 18:785–789CrossRefGoogle Scholar
  22. Parekh S, Vinei VA, Stroobel RJ (2000) Improvement of microbial strains and fermentation processes. Appl Microbiol Biotechnol 54:287–301CrossRefGoogle Scholar
  23. Phadtare SU, Deshpande VV, Srinivasan MC (1993) High activity alkaline protease from Conidiobolus coronatus (NCL 86.8.20): enzyme production and compatibility with detergents. Enzyme Microb Technol 15:72–76CrossRefGoogle Scholar
  24. Sana B, Ghosh D, Saha M, Mukherjee J (2006) Purification and characterization of a salt, solvent, detergent and bleach tolerant protease from a new gamma-Proteobacterium isolated from the marine environment of the Sundarbans. Process Biochem 41:208–215CrossRefGoogle Scholar
  25. Sharma R, Chisti Y, Banerjee UC (2001) Production, purification, characterization, and applications of lipases. Biotechnol Adv 19:627–662CrossRefGoogle Scholar
  26. Sigma DS, Mooser G (1975) Chemical studies of enzyme active sites. Annu Rev Biochem 44:889–931CrossRefGoogle Scholar
  27. Thangam BE, Rajkumar SG (2002) Purification and characterization of alkaline protease from Alcaligenes faecalis. Biotechnol Appl Biochem 35:149–154CrossRefGoogle Scholar
  28. Towatana N, Painupong A, Suntinanalert P (1999) Purification and characterization of an extracellular protease from alkaliphilic and thermophilic Bacillus sp. PS 719. J Biosci Bioeng 87:581–587CrossRefGoogle Scholar
  29. Tsuchida O, Yamagota Y, Ishizuka J, Arai J, Yamada J, Ta-keuchi M, Ichishima E (1986) An alkaline proteinase of an alkalophilic Bacillus sp. Curr Microbiol 14:7–12 CrossRefGoogle Scholar
  30. Wendhausen R, Frigato M, Fernandes P, Carvalho CCCR, Cruz A, Pinheiro HM, Cabral JMS (2005) Chrysotile as a support for the immobilization of Mycobacterium sp. NRRL B-3805 cells for the bioconversion of M-sitosterol in an organic-aqueous two- liquid phase system. J Mol Catal B: Enzymatic 32:61–65CrossRefGoogle Scholar
  31. Whittle G, Bloomfield G (1999) The site-specific integration of genetic elements may nodulate thermostable protease production. Microbiology 145:2845–2851Google Scholar
  32. Yamagata Y, Ichishima E (1989) A new alkaline proteinase with PI 2.8 from alkalophilic Bacillus species. Curr Microbiol 19:259–264 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  • Amit Gupta
    • 1
  • Babu Joseph
    • 2
  • Abin Mani
    • 2
  • George Thomas
    • 2
  1. 1.National Institute of Pharmaceutical Education and ResearchChandigarhIndia
  2. 2.College of Biotechnology and Allied SciencesAllahabad Agricultural Institute - Deemed UniversityAllahabadIndia

Personalised recommendations