Production, Purification and Optimisation of Amylase by Submerged Fermentation Using Bacillus subtilis

Authors

  • Aastha Acharya  Department of Microbiology, St. Xavier's College, Tribhuwan University, Kathmandu, Nepal
  • Amit Khanal  Department of Microbiology, St. Xavier's College, Tribhuwan University, Kathmandu, Nepal
  • Mrishal Ratna Bajracharya  Department of Microbiology, St. Xavier's College, Tribhuwan University, Kathmandu, Nepal
  • Aashish Timalsina  Department of Microbiology, St. Xavier's College, Tribhuwan University, Kathmandu, Nepal
  • Arjun Bishwokarma  Department of Microbiology, St. Xavier's College, Tribhuwan University, Kathmandu, Nepal
  • Anup Basnet  Lecturer, Department of Microbiology, St. Xavier's College, Tribhuwan University, Kathmandu, Nepal

DOI:

https://doi.org//10.32628/IJSRSET196122

Keywords:

Amylase, Bacillus spp, Production, Purification, Optimisation, Vegetations

Abstract

Amylase is abundantly present in nature. The main source of this enzyme is the microbial origin. It is known that about two-third of the industrial enzymes (amylase, protease, cellulose, penicillinase, chitinase, etc,) are produced by the Bacillus spp. The present work comprised in the identification of amylase producing Bacillus spp and exposure of the producers to various parameters for the maximum yield of the enzyme. To isolate and identify the amylase and protease producing strain, soil samples were collected from different vegetation from the altitude at 4367.35 feet above sea level. The isolates were screened and various biochemical tests and morphological observations were done to identify the isolates. The enzymes were produced by the submerged state fermentation (SmF) from the isolates and purified by dialysis. Effects of temperature, pH, and different carbon and nitrogen sources of the medium using SmF were optimized. Among 95 isolates, 36 were identified. Among the identified isolates, Bacillus subtilis produced the maximum yield and thus, it was optimized for the amylase production. The maximum amylase production was found at 42⁰C temperature, in fructose as a carbon sugar, peptone as a nitrogen source and at pH 7. Almost all the enzyme producers inhabited the roots of leguminous plants. In the present study, starch is used with the nutrient agar medium to help in cell immobilization for maximum production of amylase by strains of Bacillus. More sophisticated process of purification such as chromatography and electrophoresis will yield more enzyme as compared to the dialysis.

References

  1. R.M. Atlas and R Bartha. (2007). “Microbial Ecology Fundamentals and Applications”, 4th Edition. Pearson Education, pp 372-384.
  2. M.J. Pelczar, E.C.S. Chan and N.R. Krieg. (2011). “Microbiology and Applied Based Approach”, Third Reprint, Tata McGraw Hill Education Private Limited, pp 771-774. 
  3. T.M. Madigan and J. M. Martinko. (2006). Brock Biology of Microorganisms, Pearson Education Limited, pp 374- 594. 
  4. A. Probanza, L. García, J. A. Palomino, M. R. Ramos and G. F. J. Mañero. (2002). “ Pinus pinea, seedling growth and bacterial rhizosphere structure after inoculation with PGPR Bacillus (B. licheniformis CECT 5106 and B. pumilus CECT (5105)”. Applied Society and Ecology. 20:75–84. 
  5. J. Harvey, K. P. Keenan and A. Gilmour. (2007). Assessing biofilm formation by Listeria monocytogenes strains. Food Microbiology. 24: 380-392.
  6. N. A. Lyngwi and S. R. Joshi. (2014). Economically important Bacillus and related genera: a mini review”. Biology of useful plants and microbes.
  7. B. Pokhrel , P. Wanjaree and S. Singh.(2013). Isolation, screening and characterization of promising amylase producing bacteria from sewage enriched soil. Int J Adv Biotechnol Res. 4 (2): 286-290. 
  8. Priest FG (1977). Extracellular enzyme synthesis in the genus Bacillus. American Society for Microbiology. 41(3):711-753.
  9. Monteiro de Souza P and P. Magalhães. (2010). Application of amylase in industry – a review. Brazilian Journal of Microbiology. ISSN 15178382: 850-861. 
  10. K.S. Naidu. (2011). Characterization and purification of protease enzyme. Journal of Applied Pharmaceutical Science. 01 (03):107-112. 
  11. N. Akcan. (2011). Production of extracellular alkaline protease from Bacillus subtilis RSKK96 with solid state fermentation. Eurasia J Biosci. 5:64-72.
  12. R. Gupta, Q. Beg and S Khan. (2003). Applied Microbiology and Biotechnology. 60: 381.
  13. L.S. Devi, P. Khaund and S.R. Joshi (2010). Thermostable α amylase from natural variants of Bacillus spp. prevalent in eastern Himalayan Range. African Journal of Microbiology Research. 4(23):2534-2542. 
  14. T. P Keerthirathne, K. Ross, H. Fallowfield, and H. Whiley. (2016). A Review of Temperature, pH and Other Factors that Influence the Survival of Salmonellain Mayonnaise and Other Raw Egg Products. Pathogens.5(4):63. http://doi.org/10.3390/pathogens5040063.
  15. S. Manandhar and S. Sharma. (2013). Practical Approach to Microbiology. 3rd Edition. National Book Centre.
  16. Pandey A., Nigam P., Soccol C.R., Soccol V.T, Singh D. and Mohan R. (2000). Advances in microbial amylases. Biotechnol. Appl. Biochem. 31: 135-152.
  17. P. Bernfield. (1955). Amylases - and In: Methods in Enzymology. Academic Press. New York. USA. 1: 149–158. 
  18. D. A. Wood and H. Tristram. (1970). Journal of Bacteriology. 104:1045.
  19. I. N?me?ková, K. Solichová, P. Roubal, B. Uhrová and E. Šviráková. (2011). Methods for detection of Bacillus sp., B. cereus, and B. licheniformis in raw milk. Czech J. Food Sci. 29 (Special Issue): S55– S60. 
  20. T. Gibson and R. E. Gordon. (1974). Bacillus In Bergey's Manual of Determinative Bacteriology. 8th Edition, Williams & Wilkins, Baltimore, pp 529-550. 
  21. D. Daffonchio, N. Raddadi, M. Merabishvili, A. Cherif, L. Carmagnola, L. Brusetti and S. Borin. (2006). Strategy for identification of Bacillus cereus and Bacillus thuringienversis strains closely related to Bacillus anthracis. Applied and environmental microbiology. 72(2):1295-1301.
  22. F.A. Sharif and N.G Alaeddino?lu. A rapid and simple method for staining of the crystal protein of Bacillus thuringiensis. Journal of Industrial Microbiology. 
  23. M. R. Swain, S. Kar, G. Padmaja and R. C. Ray. (2006). Partial Characterization and Optimization of Production of Extracellular α-amylase from Bacillus subtilis Isolated from Culturable Cow Dung Microflora. Polish Journal of Microbiology. 55(4):289-296.
  24. Md. M. Hasan, L.W. Marzan, A. Hosna, A. Hakim and A.K. Azad (2007). Optimization of some fermentation conditions for the production of extracellular amylases by using Chryseobacterium and Bacillus isolates from organic kitchen wastes. Journal of Genetic Engineering and Biotechnology. 15:59–68. http://dx.doi.org/10.1016/j.jgeb.2017.02.009.
  25. L. Szilard (1960). Proc. U.S. Nat. Acad. Sci. 46:277. 
  26. K. Suribabu, T. L. Govardhan and K.P.J. Hemalatha. (2014). Influence of carbon sources on α- amylase production by Brevibacillus sp. under submerged fermentation. International Journal of Research in Engineering and Technology. 03: 02, eISSN: 2319-1163 | pISSN: 2321-7308.

International Journal of Scientific Research in Science, Engineering and Technology

Downloads

Published

2019-02-28

Issue

Volume 6, Issue 1, January-February-2019

Section

Research Articles

License

Copyright (c) IJSRSET

Creative Commons License

This work is licensed under a Creative Commons Attribution 4.0 International License.

How to Cite

[1]
Aastha Acharya, Amit Khanal, Mrishal Ratna Bajracharya, Aashish Timalsina, Arjun Bishwokarma, Anup Basnet, " Production, Purification and Optimisation of Amylase by Submerged Fermentation Using Bacillus subtilis, International Journal of Scientific Research in Science, Engineering and Technology(IJSRSET), Print ISSN : 2395-1990, Online ISSN : 2394-4099, Volume 6, Issue 1, pp.265-275, January-February-2019. Available at doi : https://doi.org/10.32628/IJSRSET196122