1 2007 Vol: 38(4). DOI: 10.1590/S1517-83822007000400034

Production of moderately halotolerant, SDS stable alkaline protease from Bacillus cereus MTCC 6840 isolated from lake Nainital, Uttaranchal state, India

A moderately cold active, extracellular alkaline protease producing bacterium was isolated from a fresh water lake. The isolate was found to be a gram-positive, rod shaped organism later identified as Bacillus cereus MTCC 6840. The bacterium produced the maximum amount of enzyme when allowed to grow for 24 h at temperature 25º and pH 9.0. Among a variety of substrates used, fructose as a carbon source and a combination of yeast extract and peptone as nitrogen source, supported the maximum protease production by the organism (120 U/ml). Fe++ and Co++ stimulated the enzyme activity whereas Ca++, Cu++, K+, Mg++ and Mn++ inhibited it to different extents. The protease was found to be highly stable in the presence of NaCl, SDS and acetone. Treatment with EDTA and PMSF resulted in the considerable loss of enzyme activity. The enzyme was found to be optimally active at pH 9.0 and temperature 20ºC.

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  1. Bhardwaj, M.; Satyanarayana, T. Extracellular highly alkaline and cold active protease of a psychrophile Bacillus sp. MLB-2. Int. Conf. Microb. Divers., University of Delhi, New Delhi (India), p.164 , (2005) .
  2. Borriss, R. Biology of enzymes. In: Rehm, H. and Reed, G. (eds). Biotechnol., Verlag Chemie, Weinheim , 35-62 (1987) .
  3. Chaloupka, J. Temperature as a factor regulating the synthesis of microbial enzymes. Microbiol. Sci., 290 86, (1985) .
  4. Chu, I.M.; Lee, C.; Li, T.S. Production and degradation of alkaline protease in batch culture of Bacillus subtilis ATCC 14416. Enz. Microb. Technol., 14 , 755-761 (1992) .
  5. Dhandapani, R.; Vijayaragavan, S. Production of a thermophilic, extracellular alkaline protease by Bacillus stearothermophilus AP-4. World J. Microbiol. Biotechnol., 10 , 33-35 (1994) .
  6. Frankena, J.; Koningstein, G.M.; Van-Verseveld, H.W.; Stouthamer, A.H. Effect of different limitation in chemostat culture on growth and production of exocellular protease by Bacillus licheniformis. Appl. Microbiol. Biotechnol., 24 , 106-112 (1986) .
  7. Fujiwara, N.; Yamamoto, K. Production of alkaline protease in a low cost medium by alkalophilic Bacillus sp. and properties of the enzyme. J. Ferment. Technol., 65 , 345-348 (1987) .
  8. Gajju, H.; Bhalla, T.C.; Agrawal, H.O. Thermostable alkaline protease from thermophilic Bacillus coagulans PB-77. Ind. J. Microbiol., 36 , 153-155 (1996) .
  9. Ghorbel-Frikha, B.; Sellami-Kamoun, A.; Fakhfakh, N.; Haddar, A.; Manni, L.; Nasri, M. Production and purification of a calcium-dependent protease from Bacillus cererus BG1. J. Ind. Microbiol. Biotechnol., 32 , 186-194 (2005) .
  10. Gupta, R.K.; Saxena R.K.; Khan, S. Bleach stable, alkaline protease from Bacillus sp. Biotechnol. Lett., 21 , 135-138 (1999) .
  11. Johnvesly, B.; Naik, G.R. Studies on production of thermostable alkaline protease from thermophilic and alkalophilic Bacillus sp. JB-99 in a chemically defined medium. Process Biochem., 37 , 139-144 (2001) .
  12. Joo, H.S.; Chang, C.S. Oxidant and SDS-stable alkaline protease from a halo-tolerant Bacillus clausii I-52: enchanced production and simple purification. J. Appl. Microbiol., 98 , 491-497 (2005) .
  13. Kaur, M.; Dhillon, S.; Choudhary, K.K.; Singh, R. Production, purification and characterization of a thermostable alkaline protease from Bacillus polymyxa. Ind. J. Microbiol., 38 , 63-67 (1998) .
  14. Kazan, D.; Denizci, A.A.; Oner, M.N.K.; Erarslan, A. Purification and characterization of a serine alkaline protease from Bacillus clausii GMBAE 42. J. Indus. Microbiol. Biotechnol., 32 , 335-344 (2005) .
  15. Khemlnitsky, Y.L.; Levashov, A.V.; Klyachko, N.L.; Martinek, K. Engineering biocatalytic systems in organic media with low water content. Enz. Micro. Technol., 10 , 710-724 (1988) .
  16. Kim, J.; Lee, S.M.; Jung, H.J. Characterization of calcium activated bifunctional peptidase of the psychrotrophic Bacillus cereus. J. Microbiol., 43 , 237-243 (2005) .
  17. Kobayashi, T.; Hakamada, Y.; Adachi, S.; Hitomi, J.; Yoshimatsue, T.; Koike, K.; Kawai, S.; Ito, S. Purification and properties of an alkaline protease from alkalophilic Bacillus sp. KSM-K16. Appl. Microbiol. Biotechnol., 43 , 473-481 (1995) .
  18. Kumar, D.; Bhalla, T.C. Purification and characterization of a small size protease from Bacillus sp. APR-4. Ind. J. Exp. Biol., 42 , 515-521 (2004) .
  19. Kumar, D.; Gajju, H.; Bhalla, T.C. Production of a thermostable protease by Bacillus sp. APR-4. A. J. Microbiol. Biotechnol. Env. Sci., 4 , 533-540 (2002) .
  20. Madan, M.; Dhillon, S.; Singh, R. Purification and characterization of alkaline protease from a mutant of Bacillus polymyxa. Ind. J. Microbiol., 42 , 155-159 (2002) .
  21. Manachini, P.L.; Fortina, M.G.; Parini, C. Thermostable alkaline protease from Bacillus thermoruber- A new species of Bacillus. J. Appl. Microbiol. Biotechnol., 28 , 409-413 (1988) .
  22. Margesin, R.; Dieplinger, H.; Hofmann, J.; Sarg, B.; Lindner, H. A cold-active extracellular metalloprotease from Pedobacter cryconitis- production and properties. Res. Microbiol., 156 , 499-505 (2005) .
  23. Ogino, H.; Watanabe, F.; Yamada, M.; Nakagawa, S.; Hirose, T.; Nogushi, A.; Yasuda, M.; Ishikawa, H. Purification and characterization of organic solvent stable protease from organic solvent tolerant Pseudomonas aeruginosa PST-01. J. Biosci. Bioengg., 87 , 61-68 (1999) .
  24. Ogino, H.; Yasui, K.; Shibtani, T.; Ashihara, T.; Ishikawa, H. Organic solvent tolerant bacterium which secretes an organic solvent stable proteolytic enzyme. Appl. Env. Microbiol., 61 , 4258-4262 (1995) .
  25. Rao, M.B.; Tanksale, A.M.; Ghatge, M.S.; Deshpande V.V. Molecular and biotechnological aspects of microbial proteases. Microbiol. Mol. Biol. Rev., 62 , 597-635 (1998) .
  26. Shi, J.S.; Wu, Q.F.; Xu, Z.H.; Tao, W.Y. Identification of psychrotrophs SYP-A producing cold-adapted protease from the No. 1 Glacier of China and study on its fermentation conditions. Wei. Sheng. Wu. Xue. Bao., 45, 258-263 , 2-3 (2005) .
  27. Singh, J.; Vohra, R.M.; Sahoo, D.K. Alkaline protease from a new obligate alkalophilic isolate of Bacillus sphaericus. Biotechnol. Lett., 21 , 921-924 (1999) .
  28. Sinha, N.; Satyanarayana, T. Alkaline protease production by thermophilic Bacillus licheniformis. Ind. J. Microbiol., 31 , 425-430 (1991) .
  29. Sumandeep; Bhusan, B.; Beg, Q.; Hoondal, G.S. Partial purification and characterization of a thermostable alkaline protease of an alkalophilic Bacillus sp. NG 27. Ind. J. Microbiol., 39 , 185-187 (1999) .
  30. Vazquez, S.C.; Coria, S.H.; MacCormack, W.P. Extracellular proteases from eight psychrotolerant Antarctic strains. Microbiol. Res., 159 , 157-166 (2004) .
  31. Ventosa, A.; Nieto, J.J.; Oren, A. Biology of moderately halophilic aerobic bacteria. Microbiol. Mol. Biol. Rev., 62 , 504-544 (1998) .