Skip to main content
SpringerLink
Log in

Thermal and chemical inactivation of phytase from rice bean (Vigna umbellata Thunb.)

  • Original Article
  • Published:
Journal of Proteins and Proteomics Aims and scope Submit manuscript

Abstract

This study aimed to evaluate the results of the thermal (50 °C) and the chemical inactivation kinetics (urea 0.1 M) of the rice bean phytase along with spectral analysis at elevated temperature (80 °C, 1 h) and in the presence of urea (8 M, 1 h). Thermal inactivation of the phytase from rice bean (Vigna umbellata), exhibited a biphasic kinetic pattern with a distinct fast and a slow phase in the loss of the activity. Bovine serum albumin showed a little protection. The ultraviolet spectrum of the heat-inactivated phytase exhibited a higher absorbance at all wavelengths in comparison to the native enzyme. The emission maxima of the native and heat-inactivated phytase were at 330 and 340 nm, respectively in the fluorescence spectrum. The intensity in the heat-treated enzyme was lower in comparison to the native enzyme. A biphasic curve was also obtained in the chemical inactivation of the phytase. The fluorescence spectrum exhibited a lower emission intensity in comparison to the native enzyme. The emission maximum of the inactivated phytase was at 335 nm. The result indicates that in the presence of urea, more tertiary structure is retained in comparison to the heat-inactivated phytase.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Abbreviations

BSA:

Bovine serum albumin

PEG:

Polyethyleneglycol

References

  • Ambasht PK, Malhotra OP, Kayastha AM (1996) Purification, characterization and steady state kinetic properties of cytosolic pyruvate kinase free of phosphoenolpyruvate phosphatase activity from germinating mung beans (Vigna radiata L.). Indian J Biochem Biophys 33:184–194

    CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Belho K, Ambasht PK (2017) Characterization of phytase isolated from rice bean cotyledons (Vigna umbellata Thunb.) with respect to influence of different factors. In: Bhattacharjee MN, Buam DL, Masharing C, Laloo BM (eds) Exploring chemistry -interface with human welfare. Eastern Book House Publishers, Guwahati, pp 157–166

    Google Scholar 

  • Belho K, Ambasht PK (2021) Immobilization of phytase from rice bean (Vigna umbellata Thunb.) on glutaraldehyde activated chitosan microspheres. J Sci Res 65:111–119

    Google Scholar 

  • Belho K, Nongpiur SR, Ambasht PK (2016) Purification and partial characterization of phytase from rice bean (Vigna umbellata Thunb.) germinated seeds. J Plant Biochem Biotechnol 25:27–30

    Article  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  Google Scholar 

  • Copeland RA (1994) Methods for protein analysis- a practical guide for laboratory protocols. Springer, Boston, pp 199–216

    Google Scholar 

  • Dixon M, Webb EC (1979) Enzymes, 3rd edn. Longman Group Ltd., London, pp 834–918

    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  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-inositol hexakisphosphate 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  Google Scholar 

  • Ha NC, Oh BC, Shin S, Kim HJ, Oh TK, Kim YO, Choi KY, Oh BH (2000) Crystal structures of a novel, thermostable phytase in partially and fully calcium-loaded states. Nat Struct Biol 7:147–153

    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  Google Scholar 

  • Hubel F, Beck E (1996) Maize root phytase-purification, characterization, and localization of enzyme activity and its putative substrate. Plant Physiol 112:1429–1436

    Article  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  Google Scholar 

  • Kayastha AM, Gupta AK (1987) An easy method to determine the kinetic parameters of biphasic reactions. Biochem Educ 15:135–135

    Article  CAS  Google Scholar 

  • Kayastha AM, Malhotra OP (1991) Phosphoenolpyruvate phosphatase from Vigna radiata L. Wilczek: properties and possible function. Plant Physiol Biochem 18:14–17

    Google Scholar 

  • Kostrewa D, Gruninger-Leitch F, D’Arcy A, Broger C, Mitchell D, Van Loon APGM (1997) Crystal structure of phytase from Aspergillus ficuum at 2.5 Å resolution. Nat Struct Biol 4:185–190

    Article  CAS  Google Scholar 

  • Laboure AM, Gagnon J, Lescure AM (1993) Purification and characterization of phytase (myo-inositolhexakis- phosphate phosphohydrolase) accumulated in maize (Zea mays) seedlings during germination. Biochem J 295:413–419

    Article  CAS  Google Scholar 

  • Lackowicz JR (1999) Principles of fluorescence spectroscopy. Kluwer Academic Plenum Publishers, New York, pp 529–575

    Book  Google Scholar 

  • Lim D, Golovan S, Forsberg CW, Jia Z (2000) Crystal structures of Escherichia coli phytase and its complex with phytate. Nat Struct Biol 7:108–113

    Article  CAS  Google Scholar 

  • Lolas GM, Markakis P (1977) The phytase of navy beans (Phaseolus vulgaris). J Food Sci 42:1094–1097

    Article  CAS  Google Scholar 

  • Lopez HW, Leenhardt F, Coudray C, Remesey C (2002) Minerals and phytic acid interactions: Is it a real problem for human nutrition? Int J Food Sci Technol 37:727–739

    Article  CAS  Google Scholar 

  • Mahajan A, Dua S (1997) Nonchemical approach for reducing anti-nutritional factors in rapeseed (Brassica campestris var. Toria) and characterization of enzyme phytase. J Agric Food Chem 45:2504–2508

    Article  CAS  Google Scholar 

  • Malhotra OP, Srivastava PK (1982) Isolation and characterization of isocitrate lyase of castor endosperm. Arch Biochem Biophys 214:164–171

    Article  CAS  Google Scholar 

  • Phillippy BQ (1998) Purification and catalytic properties of a phytase from scallion (Allium fistulosum L.) leaves. J Agric Food Chem 46:3491–3496

    Article  CAS  Google Scholar 

  • Rodriguez E, Mullaney EJ, Lei XG (2000) Expression of the Aspergillus fumigatus phytase gene in Pichia pastoris and characterization of the recombinant enzyme. Biochem Biophys Res Commun 268:373–378

    Article  CAS  Google Scholar 

  • Timofeevski SL, Aust SD (1997) Kinetics of calcium release from manganese peroxidase during thermal inactivation. Arch Biochem Biophys 342:169–175

    Article  CAS  Google Scholar 

  • Ullah AHJ, Dischinger HC (1993) Aspergillus ficuum phytase: Complete primary structure elucidation by chemical sequencing. Biochem Biophys Res Commun 192:747–753

    Article  CAS  Google Scholar 

  • Ullah AHJ, Gibson DM (1987) Extracellular phytase (EC 3.1.3.8) from Aspergillus ficuum NRRL 3135: purification and characterization. J Prep Biochem 17:63–91

    CAS  Google Scholar 

  • Ullah AHJ, Mullaney EJ (1996) Disulphide bonds are necessary for structure and activity in Aspergillus ficuum phytase. Biochem Biophys Res Commun 227:311–317

    Article  CAS  Google Scholar 

  • Ullah AHJ, Sethumadhavan K, Lei XG, Mullaney EJ (2000) Biochemical characterization of cloned Aspergillus fumigatus phytase (phyA). Biochem Biophys Res Commun 275:279–285

    Article  CAS  Google Scholar 

  • Ushasree MV, Sumayya HBS, Pandey A (2011) Adopting structural elements from intrinsically stable phytase – a promising strategy towards thermostable phytases. Indian J Biotechnol 10:458–467

    CAS  Google Scholar 

  • Wang XY, Meng FG, Zhou HM (2004a) Unfolding and inactivation during thermal denaturation of an enzyme that exhibits phytase and acid phosphatase activities. Int J Biochem Cell B 36:447–459

    Article  CAS  Google Scholar 

  • Wang XY, Meng FG, Zhou HM (2004b) The role of disulfide bonds in the conformational stability and catalytic activity of phytase. Biochem Cell Biol 82:329–334

    Article  CAS  Google Scholar 

  • Wei X, Wang X, Zhou B, Zhou H (2006) Effect of urea on activity and conformation of a glycoprotein. Tsinghua Sci Technol 11:400–407

    Article  CAS  Google Scholar 

  • Yao MZ, Zhang YH, Lu WL, Hu MQ, Wang W, Liang AH (2011) Phytases: crystal structures, protein engineering and potential biotechnology application. J Appl Microbiol 112:1–14

    Article  Google Scholar 

  • Zhao DM, Wang M, Mu XJ, Sun ML, Wang XY (2007) Screening, cloning and overexpression of Aspergillus niger phytase (phyA) in Pichia pastoris with favourable characteristics. Lett Appl Microbiol 45:522–528

    Article  CAS  Google Scholar 

Download references

Acknowledgements

KB is grateful for the financial assistance provided by UGC in the form of SRF, Maulana Azad National Fellowship. The research facilities provided in the Department through UGC DRS III is acknowledged.

Author information

Authors and Affiliations

  1. Department of Biochemistry, School of Life Sciences, North-Eastern Hill University, Shillong, Meghalaya, 793 002, India

    K. Belho & P. K. Ambasht

Authors
  1. K. Belho
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. P. K. Ambasht
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

KB conducted the experiments and prepared the draft manuscript. PKA was the mentor and finalized the manuscript in the present form.

Corresponding author

Correspondence to P. K. Ambasht.

Ethics declarations

Conflict of interest

Conflict of interest is ruled out.

Research involving human participants and/or animals

This article does not contain any of the experiments with human participants or animals.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Belho, K., Ambasht, P.K. Thermal and chemical inactivation of phytase from rice bean (Vigna umbellata Thunb.). J Proteins Proteom 12, 143–149 (2021). https://doi.org/10.1007/s42485-021-00061-2

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s42485-021-00061-2

Keywords

Search

Navigation