International Journal of Modern Science and Technology · ISSN 2456-0235

Research Article · Biological Sciences · Volume 2, Issue 3 · Mar 2017 · Pages 90–97

Ameliorating the phytate-degrading phytase production using an isolated Bacillus sp. from agricultural fields

M. Seenuvasan, J. Iyyappan, M. Anil Kumar, K. Vaithianathan

  • M. Seenuvasan: Department of Petrochemical Engineering, SVS College of Engineering, Coimbatore. India.
  • J. Iyyappan: Department of Biotechnology, Vel Tech High Tech Dr. Rangarajan Dr. Sakunthala Engineering, College, Chennai. India.
  • M. Anil Kumar: Department of Biotechnology, Madha Engineering College, Chennai. India.
  • K. Vaithianathan: Department of Biotechnology, Madha Engineering College, Chennai. India.

Abstract

Accumulation of Phosphate in environment can be reduced by activity of Phytase enzyme molecules. Phytases are the enzymes that instigate removal of phosphate form Phytic acid. An indigenous Bacillus sp. was screened from the vicinities of agricultural fields for the production of phytase enzyme when grown in phytase specific media. The enzyme was secreted extracellularly and the isolate exhibited high activities. The production media was optimized using statistical techniques and the quantified operational parameters were used to perform experiments in bench-top batch fermenter. The phytase was recovered and purified using ammonium sulphate precipitation, dialysis, and chromatographic techniques. The purified enzyme was characterized for its operational stability under varying pH, temperature, inducers. The purified phytase enzyme was optimally active at pH 5, 30°C and enzyme activity was stimulated by CuCl2. The kinetic parameters were evaluated and purified enzyme had specific activity and molecular weight of 3.49 U/mL and 45 kDa, respectively.

Keywords

Phytase; Bacillus species; Optimization; Purification; Characterization; Molecular weight

References

  1. Maurya AK, Parashar D, Satyanarayana T. Bioprocess for the product ion of recombinant HAP phytase of the thermophilic mold Sporotrichum thermophile and its structural an d biochemical characteristics. International Journal of B iological Macromolecules. 2017;94:36-44.
  2. Salome KR, Beazley MJ, Webb SM, Sobecky PA, Taillefert N. Biomineralization of U(VI) phosphate promoted by microbially -mediated phytate hydrolysis in contaminated soils. Geochimica et Cosmochimica Acta. 2017; 197:27-42.
  3. Barrientos L, Scott JJ, Murthy PP. Specificity of hydrolysis of phytic acid by alkaline phy tase f rom lily pollen. Plant Physiol. 1994;106:1489-1495.
  4. Cromwell GL. Antimicrobial and promicrobial ag ents. In: Lewis AJ, Southern LL (Editors). Swine nutrition. 2 nd edition. B oca Raton FL: CRC Press;2001;401-421.
  5. McCormick K, Walk CL, Wyatt CL, Adeola O. Phosphorus utilization response of pigs and broiler chickens to diets supplemented with antimicrobial s and phytase. Animal Nutrition , 2017; 3(1):77- 84.
  6. Rezaei M, Borbor S, Zaghari M, and Teimouri A. Effect of phytase supplementation on nutrients ava ilability and performance of broiler chicks. International Journal of Poultry Science . 2007;6(1):55-58.
  7. Remus J. Poultry and environment reap the benefits of new generation phytase. Feedtech: International Feed Production and Applied Nutrition. 2005;9:22-25.
  8. Megeed A and Tahir A. Reduction of phosphorus pollution from b roilers waste through supplementation of wheat based broilers feed with p hytase. Journal of Chemistry. 2015:67:1-3.
  9. Gujar PD, Bhavsar KP, Khire JM. Effect of phytase from Aspergillus niger on plan t growth and mineral assimilation in wheat (Triticum aestivum Linn.) and its potential for use as a soil amendment. J Sci Food Agric. 2013;93 (9):2242-2247.
  10. Mittal A, Singh G, Goyal V, Yadav A. and Kumar N. Optimization of medium components for phytase pr oduction on orange peel flour by Klebsiella sp. DB3 using response surface methodology. Innovative Romanian Food Biotechnology . 2011;9:35-44.
  11. Vohra A , Satyanarayana T. Phytase production by the yeast, Pichia anomala . Biotechnol Lett. 2001;23(7):551-554.
  12. Chanderman A, Puri AK, Permaul K, Singh S. Production, characteristics and applications of phytase from a rhizosphere isolated Enterobacter sp. ACSS , Bioprocess Biosyst Eng . 2016;39(10):1577-1587.
  13. Sneath PH A. Endospore -forming Grampositive rods and cocci . In: Bergey’s Manual of Systematic Bacteriology Williams & Wilkins., Baltimore, MD. 1986.
  14. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin ph enol reagent. J Bio Chem. 1951;193:264-275.
  15. Heinonen J K, Lahti RJ . Analytical Biochemistry. 1981;113:313-317.
  16. Vohra A, Satyanarayana T. Statistical optimization of the medium components by response surface methodology to enhance phytase production by Pichia anomala . Process Biochemistry. 2002;37:999-1004.
  17. Porto TS, Monteiro TIR, Morei ra KA, Lima-Filho JL, Silva MPC, Porto ALF, Carneiro-da-Cunha MG. Liquid–liquid extraction of an extracellular alkaline protease from fermentation broth using aqueous two -phase and reversed micelles systems. World J Microbiol Biotechnol. 2005;21:655-659.
  18. Laemmli U K. C leavage of structural proteins during the assembly of the he ad of bacteriophage T4. Nature. 1970 ;227:680- 686.
  19. Blum H, Beier H, Gross HJ. Improved silver staining of plant proteins, RNA and DNA in polyacr ylamide gels. Electrophoresis. 1987;8:93-99.
  20. Choi YM, Suh HJ, Kim JM. Purification and Properties of Extracellular Phytase from Bacillus sp. KHU -10. Journal of Protein Chemistry. 2001;20(4):287-292.