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Purification and partial characterization of phytase from rice bean (Vigna umbellata Thunb.) germinated seeds

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Abstract

Rice bean (Vigna umbellata Thunb.) phytase activity increased during germination and reached maximum at 72 h. The phytate content in seeds decreased with increase in germination time. Phytase was purified 32 fold from 72-h germinated cotyledons with final specific activity 2.22 U/mg. Native PAGE revealed a single band. On SDS PAGE, it revealed two bands with molecular mass 66 and 44 kDa. The native molecular mass was 110 kDa on size exclusion chromatography. The A280/260 ratio was 1.88. When the enzyme was excited at 295 nm, the emission maximum was observed at 330 nm. The FTIR results suggest that Lys, Tyr, Phe, Trp, Ser, Gln and Asn residues on the enzyme’s surface. The enzyme was stored at 4 °C, showed 12 % residual activity on 35th day which was improved to 53.6 and 65.7 %, respectively in the presence of additives ascorbic acid and acetaminophen. The optimum pH and temperature of enzyme were 4.0 and 40 °C, respectively. The energy of activation was 32.2 kJ/mol. The values of K m and V max were 0.197 mM and 2.35 μmol/min/mg protein, respectively with sodium phytate as substrate. Phytase showed broad substrate specificity. The k cat/K m ratio was the highest for sodium phytate.

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Fig. 1

Abbreviations

APase:

Acid phosphatase

BSA:

Bovine serum albumin

EDTA:

Ethylenediaminetetraaceticacid

FTIR:

Fourier transform Infrared Spectroscopy

PAGE:

Polyacrylamide gel electrophoresis

pNPP:

p-nitrophenylphosphate

References

  • Agostini JS, Nogueira RB, Ida EI (2010) Lowering of phytic acid content by enhancement of phytase and acid phosphatase activities during sunflower germination. Braz Arch Biol Technol 53:975–980

    Article  CAS  Google Scholar 

  • Andriotis VME, Ross JD (2003) Isolation and characterisation of phytase from dormant Corylus avellana seeds. Phytochemistry 64:689–699

    Article  CAS  PubMed  Google Scholar 

  • Bae HD, Yanke LJ, Cheng K-J, Selinger LB (1999) A novel staining method for detecting phytase activity. J Microbiol Methods 39:17–22

    Article  CAS  PubMed  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  • Brune M, Rossander-Hulten L, Hallberg L, Gleerup A, Sandberg A-S (1992) Iron absorption from bread in humans: inhibiting effects of cereal fiber, phytate and inositol phosphates with different number of phosphate groups. J Nutr 122:442–449

    CAS  PubMed  Google Scholar 

  • Duff SMG, Sarath G, Plaxton WC (1994) The role of acid phosphatases in plant phosphorus metabolism. Physiol Plant 90:791–800

    Article  CAS  Google Scholar 

  • Gibson DM, Ullah AHJ (1988) Purification and characterization of phytase from cotyledons of germinating soybean seeds. Arch Biochem Biophys 260:503–513

    Article  CAS  PubMed  Google Scholar 

  • Greiner R (2002) Purification and characterization of three phytases from germinated lupine seeds (Lupinus albus var. Amiga). J Agric Food Chem 50:6858–6864

    Article  CAS  PubMed  Google Scholar 

  • Greiner R, Alminger ML (1999) Purification and characterization of a phytate-degrading enzyme from germinated oat (Avena sativa). J Sci Food Agric 79:1453–1460

    Article  CAS  Google Scholar 

  • Greiner R, Konietzny U, Jany KD (1998) Purification and properties of a phytase from rye. J Food Biochem 22:143–161

    Article  CAS  Google Scholar 

  • Greiner R, Jany KD, Alminger ML (2000) Identification and properties of myo-inositolhexakisphosphate phosphohydrolases (Phytases) from barley (Hordeum vulgare). J Cereal Sci 31:127–139

    Article  CAS  Google Scholar 

  • Greiner R, Muzquiz M, Burbano C, Cuadrado C, Pedrosa MM, Goyoga C (2001) Purification and characterization of a phytate-degrading enzyme from germinated faba beans (Vicia faba var. Alameda). J Agric Food Chem 49:2234–2240

    Article  CAS  PubMed  Google Scholar 

  • Hara A, Ebina S, Kondo A, Funaguma T (1985) A new type of phytase from pollen of Typha latifolia L. Agric Biol Chem 49:3539–3544

    Article  CAS  Google Scholar 

  • Heinonen JK, Lahti RJ (1981) A new and convenient colorimetric determination of inorganic orthophosphate and its application to the assay of inorganic pyrophosphatase. Anal Biochem 113:313–317

    Article  CAS  PubMed  Google Scholar 

  • Jog SP, Garchow BG, Mehta BD, Murthy PPN (2005) Alkaline phytase from lily pollen: investigation of biochemical properties. Arch Biochem Biophys 440:133–140

    Article  CAS  PubMed  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  CAS  PubMed  Google Scholar 

  • Latta M, Eskin M (1980) A simple and rapid colorimetric method for phytate determination. J Agric Food Chem 29:1313–1315

    Article  Google Scholar 

  • Li MG, Osaki M, Honma M, Tadano T (1997) Purification and characterization of phytase induced in tomato roots under phosphorus-deficient conditions. Soil Sci Plant Nutr 43:179–190

    Article  CAS  Google Scholar 

  • Ornstein L, Davis BJ (1964) Disc electrophoresis, I. Background and theory. NY Acad Sci 121:321–349

    Article  CAS  Google Scholar 

  • Rothe GM (2006) In: Eisenthal R, Danson MJ (eds.) Enzyme assays. Oxford University Press Inc., New York. Chapter 8, ISBN 0-19-568316-1

Download references

Acknowledgments

Maulana Azad and Rajiv Gandhi SRF to KB and SRN are gratefully acknowledged. Authors acknowledge Head, Department of Chemistry, NEHU, Shillong (FTIR facility) and support through UGC DRS II, DST PURSE and DBT NER.

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Authors and Affiliations

  1. Department of Biochemistry, School of Life Sciences, North-Eastern Hill University, Shillong, 793 022, Meghalaya, India

    K. Belho, S. R. Nongpiur & P. K. Ambasht

Authors
  1. K. Belho
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  2. S. R. Nongpiur
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  3. P. K. Ambasht
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Corresponding author

Correspondence to P. K. Ambasht.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Table S1

Purification of phytase from the cotyledons of 72 h germinated rice bean (60 g) (DOCX 11 kb)

Table S2

Substrate specificity of rice bean phytase (DOCX 11 kb)

Table S3

Kinetic parameters of rice bean phytase on different substrates (DOCX 11 kb)

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Belho, K., Nongpiur, S.R. & Ambasht, P.K. Purification and partial characterization of phytase from rice bean (Vigna umbellata Thunb.) germinated seeds. J. Plant Biochem. Biotechnol. 25, 327–330 (2016). https://doi.org/10.1007/s13562-015-0333-7

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  • DOI: https://doi.org/10.1007/s13562-015-0333-7

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