Skip to main content
SpringerLink
Log in

Immobilization and characterization of acid phosphatase from wheat germ (Type I) in agarose gel

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

Abstract

Acid phosphatase (specific activity 1.82 U/mg) from wheat germ was immobilized by entrapment in agarose gel. Blocks of 5 × 5 mm dimension were used for characterization. The optimum percent immobilization was 75.25% when agarose–enzyme mixture concentration was 1.35%. The soluble and immobilized acid phosphatase exhibited emission maxima at 335 nm, respectively, when excitation wavelength was 280 nm. The intensity of immobilized enzyme was lower than corresponding soluble enzyme. Fourier Transform Infrared spectrum exhibited bands at wave numbers 3282.2 and 1635.9 cm−1 for immobilized enzyme. The optimum pH and temperature for immobilized acid phosphatase were 5.5 and 60 °C, respectively. The Km values for soluble and immobilized acid phosphatase were 0.068 and 0.232 mM, respectively. The Vmax values for soluble and immobilized acid phosphatase were 1.92 and 2.34 µmol/min/mg protein, respectively. The immobilized enzyme retained 48% activity on 56th day when stored at 4 °C. The same enzyme blocks could be reused up to 8 cycles. The method of immobilization reported here is simple and cost effective and will be suitable for further applications.

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

APase:

Acid phosphatase

FTIR:

Fourier transform infrared

p-NPP:

p-Nitrophenylphosphate

References

  • Allen SH, Nuttleman PR, Ketcham CM, Roberts RM (1989) Purification and characterization of human bone tartrate-resistant acid phosphatase. J Bone Miner Res 4:47–55

    CAS  PubMed  Google Scholar 

  • Almeida AFd, Terrasan CRF, Terrone CC, Tauk-Tornisielo SM, Carmona EC (2018) Biochemical properties of free and immobilized Candida viswanathii lipase on octyl-agarose support. Hydrolysis of triacylglycerol and soy lecithin. Process Biochem 65:71–80

    CAS  Google Scholar 

  • Ambasht PK, Kayastha AM (1999) Plant phosphoenolpyruvate phosphatase: a review. Physiol Mol Biol Plants 5:1–6

    Google Scholar 

  • Bagal D, Karve MS (2006) Entrapment of plant invertase within novel composite of agarose-guar gum biopolymer membrane. Anal Chim Acta 555:316–321

    CAS  Google Scholar 

  • Belho K, Nonpiur SR, Ambasht PK (2014) Immobilization of acid phosphatase (Type I) from wheat germ on glutaraldehyde activated chitosan beads: optimization and characterization. J Proteins Proteom 4:177–183

    Google Scholar 

  • Belho K, Nonpiur SR, Ambasht PK (2016) Purification and partial characterization of phytase from rice bean (Vigna umbellata Thunb.) germinated seeds. J Plant Biochem Biotechnol 25:327–330

    CAS  Google Scholar 

  • Biswas TK, Cundiff C (1991) Multiple forms of acid phosphatases in germinating seeds of Vigna sinensis. Phytochemistry 41:1457–1458

    Google Scholar 

  • Bolivar JM, Wilson L, Ferrarotti SA, Guisan JM, Fernandez-Lafuente R, Mateo C (2006) Improvement of the stability of alcohol dehydrogenase by covalent immobilization on glyoxyl-agarose. J Biotechnol 125:85–94

    CAS  PubMed  Google Scholar 

  • Bolivar JM, Lopez-Gallego F, Godoy C, Rodrigues DS, Rodrigues RC, Batalla P, Rocha-Martin J, Mateo C, Giordano RLC, Guisan JM (2009) The presence of thiolated compounds allows the immobilization of enzymes on glyoxyl-agarose at mild pH values: new strategies of stabilization by multipoint covalent attachment. Enzyme Microb Technol 45:477–483

    CAS  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

    CAS  PubMed  Google Scholar 

  • Bull H, Murray PG, Thomas D, Fraser AM, Nelson PN (2002) Acid phosphatase. J Clin Pathol Mol Pathol 55:65–72

    CAS  Google Scholar 

  • Chang M-Y, Juang R-S (2004) Stability and catalytic kinetics of acid phosphatase immobilized on composite beads of chitosan and activated clay. Process Biochem 39:1087–1091

    CAS  Google Scholar 

  • Chang M-Y, Juang R-S (2007) Stability and reactivity of acid phosphatase immobilized on composite beads of chitosan and ZrO2 powders. Int J Biol Macromol 40:224–231

    CAS  PubMed  Google Scholar 

  • Dixon M, Webb EC, Thorne CJR, Tipton KF (1979) Enzymes, 3rd edn. Longman Group Ltd., London

    Google Scholar 

  • dos Santos JCS, Rueda N, Torres R, Barbosa O, Goncalves LRB, Fernandez-Lafuente R (2015) Evaluation of divinylsulfone activated agarose to immobilize lipase and to tune their catalytic properties. Process Biochem 50:918–927

    Google Scholar 

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

    CAS  Google Scholar 

  • Dutta S, Christena LR, Rajaram YRS (2013) Enzyme immobilization: an overview on techniques and support materials. 3 Biotech 3:1–9

    Google Scholar 

  • Elnashar MMM (2010) Review article: immobilized molecules using biomaterials and nanobiotechnology. J Biomater Nanobiotechnol 1:61–77

    CAS  Google Scholar 

  • Fernandez-Lopez L, Rueda N, Bartolome-Cabrero R, Rodriguez MD, Albuquerque TL, dos Santos JCS, Barbosa O, Fernandez-Lafunte R (2016) Improved immobilization and stabilization of lipase from Rhizomucor miehei on octyl-glyoxyl agarose beads by using CaCl2. Process Biochem 51:48–52

    CAS  Google Scholar 

  • Ferreira CV, Granjeiro JM, Taga EM, Aoyama H (1998) Purification and characterization of multiple forms of soybean seed acid phosphatases. Plant Physiol Biochem 36:487–494

    CAS  Google Scholar 

  • Gellatly KS, Moorhead GBG, Duff SMG, Lefebvre DD, Plaxton WC (1994) Purification and characterization of potato tuber acid phosphatase having significant phosphotyrosine phosphatase activity. Plant Physiol 106:223–232

    CAS  PubMed  PubMed Central  Google Scholar 

  • Goldstein L (1976) Kinetic behavior of immobilized enzyme systems. Methods Enzymol 44:397–443

    CAS  PubMed  Google Scholar 

  • Granjeiro PA, Cavagis ADM, Leite LDC, Ferreira CV, Granjeiro JM, Aoyama H (2004) The thermal stability of castor bean acid phosphatase. Mol Cell Biochem 266:11–15

    CAS  PubMed  Google Scholar 

  • Guerrero C, Vera C, Serna N, Illanes A (2017) Immobilization of Aspergillus oryzae β-galactosidase in an agarose matrix functionalized by four different methods and application to the synthesis of lactulose. Bioresour Technol 232:53–63

    CAS  PubMed  Google Scholar 

  • Homma T, Kondo M, Kuwahara T, Shimomura M (2014) Bio- and bioelectro-catalytic properties of polyaniline/poly (acrylic acid) composite films bearing covalently-immobilized acid phosphatase. React Funct Polym 74:31–36

    CAS  Google Scholar 

  • Huang Q, Liang W, Cai P (2005) Adsorption, desorption and activities of acid phosphatase on various colloidal particles from an Ultisol. Colloid Surf B 45:209–214

    CAS  Google Scholar 

  • Hussain WM, Feder D, Schenk G, Guddat LW, McGeary RP (2018) Purple acid phosphatase inhibitors as leads for osteoporosis chemotherapeutics. Eur J Med Chem 157:462–479

    Google Scholar 

  • Juang R-S, Wu F-C, Tseng R-L (2001) Solute adsorption and enzyme immobilization on chitosan beads prepared from shrimp shell wastes. Bioresour Technol 80:187–193

    CAS  PubMed  Google Scholar 

  • Ketchman CM, Baumbach GA, Bazer FW, Roberts RM (1985) The type 5 acid phosphatase from spleen of humans with hairy cell leukemia. J Biol Chem 260:5768–5776

    Google Scholar 

  • Klabunde T, Strater N, Frohlich R, Witzel H, Krebs B (1996) Mechanism of Fe(III)–Zn (II) purple acid phosphatase based on crystal structures. J Mol Biol 259:737–748

    CAS  PubMed  Google Scholar 

  • Kurita K, Yoshino H, Nishimura S-I, Ishii S, Mori T, Nishiyama Y (1977) Mercapto-chitins: a new type of supports for effective immobilization of acid phosphatase. Carbohydr Polym 32:171–175

    Google Scholar 

  • Pedroche J, Yust MDM, Mateo C, Fernandez-Lafunte R, Giron-Calle J, Alaiz M, Vioque J, Guisan JM, Millan F (2007) Effect of the support and experimental conditions in the intensity of the multipoint covalent attachment of proteins on glyoxyl-agarose supports: correlation between enzyme-support linkages and thermal stability. Enzyme Microb Technol 40:1160–1166

    CAS  Google Scholar 

  • Plaxton WC (1996) The organization and regulation of plant glycolysis. Annu Rev Plant Physiol Plant Mol Biol 47:185–214

    CAS  PubMed  Google Scholar 

  • Poonkuzhali K, Palvannan T (2013) Comparison of biopolymers for immobilization of laccase: eco-toxicity assessment of azo dye. Indian J Biotechnol 12:395–401

    CAS  Google Scholar 

  • Prakash O, Jaiswal N (2011) Immobilization of thermostable α-amylase on the agarose and agar matrices and its application in starch stain removal. World Appl Sci J 13:572–577

    CAS  Google Scholar 

  • Reddy KRC, Kayastha AM (2006) Improved stability of urease upon coupling to alkylamine and arylamine glass and its analytical use. J Mol Catal B Enzym 38:104–112

    CAS  Google Scholar 

  • Schenk G, Ge Y, Carrington LE, Wynne CJ, Searle IR, Caroll BJ, Hamilton S, de Jersey J (1999) Binuclear metal centres in plant purple acid phosphatases: Fe-Mn in sweet potato and Fe-Zn in soybean. Arch Biochem Biophys 370:183–189

    CAS  PubMed  Google Scholar 

  • Schenk G, Mitic N, Hanson GR, Combo P (2013) Purple acid phosphatase: a journey into the function and mechanism of a colourful enzyme. Coord Chem Rev 257:473–482

    CAS  Google Scholar 

  • Sheffield DJ, Harry TR, Smith AJ, Rogers LJ (1995) Corallina officinalis bromoperoxidase immobilized on agarose. Phytochemistry 38:1103–1107

    CAS  Google Scholar 

  • Siar E-H, Zaak H, Kornecki JF, Zidoune MN, Barbosa O, Fernandez-Lafunte R (2017) Stabilization of ficin extract by immobilization on glyoxyl-agarose. Preliminary characterization of the biocatalyst performance in hydrolysis of proteins. Process Biochem 58:98–104

    CAS  Google Scholar 

  • Srivastava PK, Anand A (2014) Immobilization of acid phosphatase from Vigna aconitifolia seeds on chitosan beads and its characterization. Int J Biol Macromol 64:150–154

    CAS  PubMed  Google Scholar 

  • Tardioli PW, Vieira MF, Vieira AMS, Zanin SM, Betancor L, Mateo C, Fernandez-Lorente G, Guisan JM (2011) Immobilization–stabilization of glucoamylase: chemical modification of the enzyme surface followed by covalent attachment on highly activated glyoxyl-agarose supports. Process Biochem 46:409–412

    CAS  Google Scholar 

  • Vincent JB, Crowder MW, Averill BA (1992) Hydrolysis of phosphate monoesters: a biological problem with multiple chemical solutions. Trends Biochem Sci 17:105–110

    CAS  PubMed  Google Scholar 

  • Wang R, Cai Q, Tong D, Nie L, Yao S (1998) Enzymatic analysis of acid phosphatase and microanalysis of Cu++ and Ag+ with a SAW-impedence sensor. Enzyme Microb Technol 22:36–43

    CAS  Google Scholar 

  • Yamato S, Kawakami N, Shimada K, Ono M, Idei N, Itoh Y (2000) Preparation and characterization of immobilized acid phosphatase used for an enzyme reactor: evaluation in flow-injection analysis and pre column liquid chromatography. Anal Chem Acta 406:191–199

    CAS  Google Scholar 

  • Zaak H, Siar E-H, Kornecki JF, Fernandez-Lopez L, Pedrero SG, Virgen-Ortiz JJ, Fernandez-Lafuente R (2017) Effect of immobilization rate and enzyme crowding on enzyme stability under different conditions. The case of lipase from Thermomyces lanuginosus immobilized on octyl agarose beads. Process Biochem 56:117–123

    CAS  Google Scholar 

  • Zhu J, Huang Q, Pigna M, Violante A (2010) Immobilization of acid phosphatase on uncalcined and calcined Mg/Al-CO3 layered double hydroxides. Colloid Surf B 77:166–173

    CAS  Google Scholar 

Download references

Acknowledgements

The research facilities provided in the Department through UGC DRS III and DBT NER is acknowledged. Ms. Tutu Kalita is grateful to UGC for research fellowship in the form of JRF and SRF. The authors are grateful to Head, Department of Chemistry, NEHU, for providing FTIR facility and Prof. Ghanshyam Bez, Department of Chemistry, NEHU for useful discussions.

Author information

Authors and Affiliations

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

    Tutu Kalita & P. K. Ambasht

Authors
  1. Tutu Kalita
    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

TK conducted the experiments and was involved in writing the manuscript. PKA was the mentor, conceived the ideas, wrote discussion and shaped the overall manuscript.

Corresponding author

Correspondence to P. K. Ambasht.

Ethics declarations

Conflict of interest

The authors do not have any conflict of interest.

Research involving human participants and/or animals

This article does not contain any studies 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

Kalita, T., Ambasht, P.K. Immobilization and characterization of acid phosphatase from wheat germ (Type I) in agarose gel. J Proteins Proteom 10, 291–297 (2019). https://doi.org/10.1007/s42485-019-00023-9

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s42485-019-00023-9

Keywords

Search

Navigation