9th International Science Congress (ISC-2019).  International E-publication: Publish Projects, Dissertation, Theses, Books, Souvenir, Conference Proceeding with ISBN.  International E-Bulletin: Information/News regarding: Academics and Research

Optimization studies of Protease enzyme in in-vitro conditions from Bacillus licheniformis

Author Affiliations

  • 1Sangenomics Research Labs, Domlur layout, Bangalore, INDIA
  • 2 Department of Microbiology, Jain University, Bangalore, INDIA

Int. Res. J. Biological Sci., Volume 3, Issue (2), Pages 34-39, February,10 (2014)


Protease enzymes are of immense commercial value and find its applications in various industrial sectors. Microbes serve as a major source for proteases enzyme especially Bacillus strains. In this study protease producing bacteria was isolated from the soil sample. Screening was done on skim milk casein agar media. The colony with the highest protease production was used as the test organisms and maintained on the nutrient agar slants. The isolated protease producing microorganism was characterized morphologically and biochemically by Bergey’s Manual of Determinative Bacteriology and identified to be as Bacillus licheniformis. Optimization studies were taken up with two physical and two chemical parameters. The organism showed maximum enzyme production at pH of 8.0, temperature 37°C, with 1.5% carbon source and 1.0% nitrogen source. Mass production was carried out for the protease enzyme with optimized media parameters and purified by centrifugation, ammonium sulphate precipitation (salting out), dialysis and Sephadex G-100 gel chromatography. The enzyme activity of the purified protease was assayed to be 6.66 U/ml. The 12% SDS-PAGE has revealed a unique linear band with a molecular weight of 52-53kDa. The results of this study revealed that the bacterial strain Bacillus licheniformis is a potent source for protease enzyme.


  1. Gupta R. Beg Q. K. and Lorenz P., Bacterial alkaline proteases: molecular approaches and industrial applications, Applied Microbiology and Biotechnology, 59 (1), 15-32 (2003)
  2. Godfrey T. and West. S., 1996. Textiles: in Industrial enzymology, 2 ed., Macmillan Publishers., New York, N.Y., pp: 3.
  3. Vamsi Krishna K. Mayank Gupta. Nikhil Gupta. Hipal Gaudani. Soham Trivedi. Prasad Patil. Girish Gupta. Yogesh Khairnar. Amol Borasate. and Dharmendra Mishra., Optimization of growth and production of protease by Penicillium species using submerged fermentation, International Journal of Microbiology Research., 1(1), pp-14-18 (2009)
  4. Mehrato S. Pandey PK. Gaur R. and Darmwal N.S., The production of alkaline protease by a Bacillus species isolate, Bioresour. Technol., 67, 201-203 (1999)
  5. Gessesse A. Hatti-Kaul R. Gashe B. and Mattason B., Novel alkaline proteases from alkaliphilic bacteria grown using chicken feather, Enz. Microbiol. Technol.,32, 519-524 (2003)
  6. Sadia A. Abdul H. Dennis S. and Priya M., Purification and characterization of a novel protease from Bacillus strain SAL1, Afr. J. Biotechnol., 8(15), 3603-360 (2009)
  7. Shirish R. and Dabholkar P. S., Production of protease by Aspergillus sp. using solidstate fermentation, Afr. J. Biotechnol., 8(17), 4197-4198 (2009)
  8. Ibrahim Noor Azlina and Yusoff Norazila., Thermostable Alkaline Serine Protease from Thermophilic Bacillus Species, International Research Journal of Biological Sciences., 2(2), 29-33 (2013)
  9. Boonyanas S. Supachok S. Suree P. and Shuitein C., Purification and characterization of the highly thermostable proteases from Bacillus stearothermophilus TLS33, Protein Exp. Purif., 20, 142-151 (2000)
  10. Gabriela O. Slawomir D. and Jozef K., High-level expression, secretion, and purification of the thermostable aqualysin I from Thermus aquaticus YT-1 in Pichia pastoris, Protein Exp. Purif ., 29, 223-229 (2003)
  11. Ferrero M.A. Castro G. R. Abate C. M. Baigori M. D. and Singeriz F., Thermostable alkaline proteasesof Bacillus licheniformis MIR 29: isolation, production and characterization, Appl. Microbiol. Biotechnol.,45, 327-332 (1996)
  12. Naidu K.S.B. and Devi K.L., Optimization of thermostable alkaline protease production from species of Bacillus using rice bran, Afr. J. Biotechnol., 724-726 (2005)
  13. Sen S. and Satyanarayana T., Optimization of alkaline protease production by thermophilic Bacillus licheniformis S-40, Indian. J. Microbiol., 33, 43-47 (1993)
  14. Horikoshii K., Extracellular enzymes In Horikoshii K (Ed), Alkaliphiles Harwood Acad Pub Japan., 147-285 (1999)
  15. Banerjee U.C. Sani R. K. Azmi W. and Soni R., Thermostable alkaline protease from Bacillus brevis and its characterization as a laundry detergent additive, Proc. Biochem., 35, 213-219 (1999)
  16. Bholay A.D. More S.Y. Patil V.B. and Patil Niranjan., Bacterial Extracellular Alkaline Proteases and its Industrial Applications, International Research Journal of Biological Sciences., 1(7), 1-5 (2012)
  17. Negi S. and Banerjee R., Optimization of amylase and protease production from Aspergillus awamoriin single bioreactor through EVOP factorial design technique, Food Technol Biotechnol., 44, 257–61 (2006)
  18. Holt J.G. Krieg N. R. Sneath P.H.A. Staley J.T. and Williams S.T., Bergey's Manual of Determinative Bacteriology, 9th Edition, Williams and Wilkins USA, 559-565 (1994)
  19. Folin O. and Ciocalteau V., Enzymatic Assay of Protease Using Casein as a Substrate, J. Biol. Chem., 73, 627 (1929)
  20. Soundra Josephine F, Ramya V.S. Neelam Devi. Suresh Babu Ganapa. Siddalingeshwara K. G. Venugopal N. and Vishwanatha T., Isolation, production and characterization of protease from Bacillus Sp isolated from soil sample, J. Microbiol. Biotech. Res.,2 (1),163-168 (2012)
  21. Laemmli U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature Lond., 227, 680-685 (1970)
  22. Rahman R.N.Z.A., Basri L.P.G.M., Salleh A.B., Physical factors affecting the production of organic solvent-tolerant protease by Pseudomonas aeruginosa strain K, Bioresource. Technol., 96, 429-436 (2005)
  23. Kawai S. and Ikeda S., Studies on digestive enzymes of fishes. Effect of dietary change on the activities of digestive enzymes in carp intestine, Bull. Jpn. Soc. Sci. Fish., 38, 265-270 (1972)
  24. Folasade Olajuyigbe M. and Joshua Ajele O., Some Properties of Extracellular Protease from Bacillus licheniformisLbbl-11 Isolated from “iru”, A Traditionally Fermented African Locust Bean Condiment, Global Journal of Biotechnology & Biochemistry.,3 (1), 42-46 (2008)
  25. Udandi Boominadhan. and Rajendran Rajakumar., Optimization of Protease Enzyme Production Using Bacillus Sp. Isolated from Different Wastes, Botany Research International.,2 (2), 83-87 (2009)
  26. Adinarayana K. and Elliah P., Response surface optimization of the critical medium component for the production of alkaline protease by a newly isolated Bacillus sp, J Pharm Pharmaceut Sci., 272-278 (2002)
  27. Muthu Padmapriya. and Christudhas Williams B., Purification and characterization of neutral protease enzyme from Bacillus Subtilis, J. Microbiol. Biotech. Res., 2 (4), 612-618 (2012)
  28. Nihan Sevinc. and Elif Demirkan., Production of Protease by Bacillus sp. N-40 Isolated from Soil and Its Enzymatic Properties, J. Biol. Environ. Sci., 5(14), 95-103 (2011)