Purification and properties of a phytase from Candida melibiosica 2491

  • Danail Georgiev University of Plovdiv, Faculty of Biology, Dept. of Microbiology, 24 Tsar Ivan Asen Str., Plovdiv 4000, Bulgaria
  • Georgi Dobrev University of food technology, Department of Biochemistry and Molecular Biology, 26 Maritsa Blvd., Plovdiv 4000, Bulgaria
  • Stefan Shilev Agricultural University – Plovdiv, Department of Microbiology and Environmental Biotechnologies, 12 Mendeleev Str., Plovdiv 4000, Bulgaria

Abstract

Aim: To characterize the enzyme phytase produced by phytase-active Candida melibiosica 2491 for subsecuent use in feed industry. Methods: C. melibiosica 2491 had been selected among 118 strains as the most productive strain of phytase. In present study, the enzyme was first purified through electrophoresis grade in four steps: precipitation with organic solvent, ultrafiltration, gel chromatography and Denaturing gel electrophoresis (SDS–PAGE). Results: Higher levels of purification were obtained using ethanol. The gel chromatography showed an elution maximum at 11-12 fractions that characterize the corresponding one as high-molecular weight phytase. The purification level was found to be 19.5 folds with specific enzyme activity of 2.75 U/mg protein and yield – 19.64 %. Furthermore, the molecular weight of purified phytase was estimated to 35.9 кDa, with optimum of pH – at 4.5 and optimum of temperature at 55 °C. Maximum phytase activity in case of whole cells was found at 50 оС, which was less than using the purified enzyme. It was activated through 5 mM of Ba2+, 10 mM of Mn2+ and K+ ions. Total inhibition effect was achieved from Fe3+, Hg2+ and Zn2+. Copper ions (Cu2+) in concentrations at 5 mM conducted to partial inhibition effect, but at 10 mM the phytase activity was equal to zero. Low inhibition effect was determined in case of cobalt ions (Co2+) at concentrations of 10 mM. The phytase displayed broad sub­strate specificity and the Km for phytate was estimated to be 0.21 mM under the experimental conditions, while Vmax – 19.9 µМ/ml. Conclusion: Although the phytase produced by C. melibiosica 2491 is a promising enzyme to be used successfully in feed production, more investigations are needed to ensure its advantages.

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How to Cite
Georgiev, D., G. Dobrev, and S. Shilev. “Purification and Properties of a Phytase from Candida Melibiosica 2491”. Emirates Journal of Food and Agriculture, Vol. 30, no. 11, Dec. 2018, pp. 927-34, doi:https://doi.org/10.9755/ejfa.2018.v30.i11.1857. Accessed 23 Oct. 2021.
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Regular Articles