Skip to main content
SpringerLink
Log in

A three step approach for the purification of alkaline phosphatase from non-pasteurized milk

  • Short Communication
  • Published:
Journal of Food Science and Technology Aims and scope Submit manuscript

Abstract

In this study, a three step purification of alkaline phosphatase from non-pasteurized milk has been described. It included cream extraction, n-butanol treatment and acetone precipitation. Different parameters such as buffer concentration, temperature, pH, substrate concentration, acetone and n-butanol treatment were optimized to maximize the enzyme activity. The enzyme was fruitfully purified up to homogeneity from the milk, with percentage recovery and fold purification of 56.17 and 17.67 respectively. The kinetic parameters were determined to be 0.927 mM (Km) and 55.86 μM/min (Vmax), with specific activity of 11.31 U/mg. Other optimized parameters were estimated as a buffer concentration of 0.5 M with pH 9.0, temperature optima at 37 °C, with n-butanol and acetone concentration of 20 % (v/v) and 50 % (v/v) respectively. This approach provides a simple and effective method for the purification of alkaline phosphatase from non-pasteurized milk.

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
Fig. 7

References

  • Ahn YS, Snow LD (1993) Selective extraction of alkaline phosphatase and 5′-nucleotidase from milk fat globule membranes by a single phase n-butanol procedure. Prep Biochem 23:409–419

    CAS  Google Scholar 

  • Albillos SM, Reddy R, Salter R (2011) Evaluation of alkaline phosphatase detection in dairy products using a modified rapid chemiluminescent method and official methods. J Food Prot 74:1144–1154. doi:10.4315/0362-028X.JFP-10-422

    Article  CAS  Google Scholar 

  • Atlan D, Portalier R (1987) Purification of extracellular alkaline phosphatase released by Escherichia coli excretory mutants. Appl Microbiol Biotechnol. doi:10.1007/BF00256661

    Google Scholar 

  • Chaudhuri G, Chatterjee S, Venu-Babu P et al (2013) Kinetic behaviour of calf intestinal alkaline phosphatase with pNPP. Indian J Biochem Biophys 50:64–71

    CAS  Google Scholar 

  • Dean RL (2002) Kinetic studies with alkaline phosphatase in the presence and absence of inhibitors and divalent cations. Biochem Mol Biol Educ 30:401–407. doi:10.1002/bmb.2002.494030060138

    Article  CAS  Google Scholar 

  • Fadiloglu S, Erkmen O, Sekeroglu G (2004) Thermal and carbon dioxide inactivation of alkaline phosphatase in buffer and milk. Food Technol Biotechnol 42:27–32

    CAS  Google Scholar 

  • Hethey J, Lai J, Loutet S et al (2002) Effects of tricine, glycine and tris buffers on alkaline phosphatase activity. J Exp Microbiol Immunol 2:33–38

    Google Scholar 

  • Junior AB, GuimarAes LHS, Terenzi HF et al (2008) Purification and biochemical characterization of thermostable alkaline phosphatases produced by Rhizopus microsporus var. rhizopodiformis. Folia Microbiol (Praha) 53:509–516. doi:10.1007/s12223-008-0080-4

    Article  Google Scholar 

  • Kopetzki E, Lehnert K, Buckel P (1994) Enzymes in diagnostics: achievements and possibilities of recombinant DNA technology. Clin Chem 40:688–704

    CAS  Google Scholar 

  • Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275

    CAS  Google Scholar 

  • Mobley DM, Chengappa MM, Kadel WL, Stuart JG (1984) Effect of pH, temperature and media on acid and alkaline phosphatase activity in “clinical” and “nonclinical” isolates of Bordetella bronchiseptica. Can J Comp Med 48:175–178

    CAS  Google Scholar 

  • Morton RK (1953a) Alkaline phosphatase of milk. 1. Association of the enzyme with a particulate lipoprotein complex. Biochem J 55:786–795

    CAS  Google Scholar 

  • Morton RK (1953b) Alkaline phosphatase of milk. 2. Purification of the enzyme. Biochem J 55:795–800

    CAS  Google Scholar 

  • Munson L, Fall RR (1978) Purification and characterisation of Escherichia coli alkaline phosphatase. A biochemical experiment. Biochem Educ 6:53–56. doi:10.1016/0307-4412(78)90059-6

    Article  CAS  Google Scholar 

  • Neumann H, Wilson KJ, Hauck-Granoth R, Haran-Ghera N (1971) A Comparative Biochemical Study of Alkaline Phosphatases in Normal and Leukemic Mice. Cancer Res 31:1695–1701

    CAS  Google Scholar 

  • Njoku V, Chikezie P, Kaoje A (2013) Kinetic studies of alkaline phosphatase extracted from rabbit (Lepus townsendii) liver. African J Biotechnol 10:3157–3162. doi:10.4314/ajb.v10i16

    Google Scholar 

  • Thompson LMM, Robert A (1974) Factors affecting the activity and stability of alkaline phosphatase in a marine pseudomonad factors affecting the activity and stability of alkaline phosphatase in a marine pseudomonad. J Bacteriol 117:813–818

    CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biotechnology, National Institute of Technology, Raipur, 492010, India

    Lata Sheo Bachan Upadhyay & Nishant Verma

Authors
  1. Lata Sheo Bachan Upadhyay
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nishant Verma
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Lata Sheo Bachan Upadhyay.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Upadhyay, L.S.B., Verma, N. A three step approach for the purification of alkaline phosphatase from non-pasteurized milk. J Food Sci Technol 52, 3140–3146 (2015). https://doi.org/10.1007/s13197-014-1578-1

Download citation

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13197-014-1578-1

Keywords

Search

Navigation