Skip to main content
SpringerLink
Log in

Entrapment of enzyme in the presence of proline: effective approach to enhance activity and stability of horseradish peroxidase

  • Original Article
  • Published:
3 Biotech Aims and scope Submit manuscript

Abstract

In this report, activity and stability of horseradish peroxidase (HRP) entrapped in polyacrylamide gel in the presence of proline (HEP) are compared with that of enzyme entrapped in absence of proline (HE). Within polyacrylamide (8%) beads, 80% entrapment yield for peroxidase was observed in the presence as well as absence of proline. The HEP (1.5 M proline) showed 170% higher activity compared to HE. HEP also showed significant increase in KM, Vmax and Kcat. At 8th cycle of use, HEP retained 40% of its activity, whereas HE retained only 10% of activity. In addition, in comparison with HE, HEP showed increased storage stability and thermo-stability. HEP showed higher activity compared to HE over an extensive range of pH (4–8), temperature (30–80 °C) and inhibitors such as NaN3, Cd2+ and Pb2+. Our results suggest that peroxidase entrapment in polyacrylamide gel in the presence of proline can be a useful approach for increasing activity and stability of horseradish peroxidase.

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

Similar content being viewed by others

Abbreviations

HE:

Horseradish peroxidase enzyme entrapped in the absence of proline

HRP:

Horseradish peroxidase enzyme

HEP:

Horseradish peroxidase enzyme entrapped in polyacrylamide gel in the presence of proline

K cat :

Turnover number, a constant which depicts the rate of turnover of an enzyme–substrate complex into product

K M :

Michaelis constant, a constant which depicts substrate concentration that allows the enzyme to attain half Vmax

V max :

Maximum rate of reaction, reaction rate when the enzyme is saturated with substrate

References

  • Agarwal S, Gupta KK, Chaturvedi VK, Kushwaha A, Chaurasia PK, Singh MP (2018) The potential application of peroxidase enzyme for the treatment of industry wastes. In: Bharati SL, Chaurasia PK (eds) Research advancements in pharmaceutical, nutritional, and industrial enzymology. IGI Global, USA, pp 278–293

    Google Scholar 

  • Agostini E, Hernandez-Ruiz J, Arnao M, Milrand SR, Tigier HA, Acosta MA (2002) A peroxidase isoenzyme secreted by turnip (Brassica napus) hairy-root cultures: inactivation by hydrogen peroxide and application in diagnostic kits. Biotechnol Appl Biochem 35:1–7

    CAS  PubMed  Google Scholar 

  • Alemzadeh I, Nejati S (2009) Phenols removal by immobilized horseradish peroxidase. J Hazard Mater 166:1082–1086

    CAS  PubMed  Google Scholar 

  • Altikatoglu M, Basaran Y (2011) Additive effect of dextrans on the stability of horseradish peroxidase. Protein J 30:84–90

    CAS  PubMed  Google Scholar 

  • Bansal N, Kanwar SS (2013) Peroxidase(s) in environment protection. Sci World J. https://doi.org/10.1155/2013/714639

    Article  Google Scholar 

  • Bayramoglu G, Arıca MY (2008) Enzymatic removal of phenol and p-chlorophenol in enzyme reactor: horseradish peroxidase immobilized on magnetic beads. J Hazard Mater 156:148–155

    CAS  PubMed  Google Scholar 

  • Bibi Z, Shahid F, Qader SAU, Aman A (2015) Agar–agar entrapment increases the stability of endo-β-1,4-xylanase for repeated biodegradation of xylan. Int J Biol Macromol 75:121–127

    CAS  PubMed  Google Scholar 

  • Bozorgmehr MR, Monhemi H (2015) How can a free amino acid stabilize a protein? Insights from molecular dynamics simulation. J Sol Chem 44:45–53

    CAS  Google Scholar 

  • Brennan JD, Benjamin D, Dibattista EM, Gulcev D (2003) Using sugar and amino acid additives to stabilize enzymes within sol–gel derived silica. Chem Mater 15:737–745

    CAS  Google Scholar 

  • Bruździak P, Adamczak B, Kaczkowska E, Czub J, Stangret J (2015) Are stabilizing osmolytes preferentially excluded from the protein surface? FTIR and MD studies. Phys Chem Chem Phys 17:23155–23164

    PubMed  Google Scholar 

  • Chiong T, Lau SY, Khor EH, Danquah MK (2014) Enzymatic approach to phenol removal from wastewater using peroxidases. OA Biotechnol 10:3–9

    Google Scholar 

  • Egley GH, Paul RN Jr, Vaughn KC, Duke SO (1983) Role of peroxidase in the development of water-impermeable seed coats in Sida spinosa L. Planta 157:224–232

    CAS  PubMed  Google Scholar 

  • Fernandez-Lafuente R (2009) Stabilization of multimeric enzymes: strategies to prevent subunit dissociation. Enzyme Microb Technol 45:405–418

    CAS  Google Scholar 

  • Garrett RH, Grisham CM (1999) Biochemistry, 2nd edn. Saunders College Publisher, Fort Worth, p 1127

    Google Scholar 

  • Husain Q, Ulber R (2011) Immobilized peroxidase as a valuable tool in the remediation of aromatic pollutants and xenobiotic compounds: a review. Crit Rev Environ Sci Technol 41:770–804

    CAS  Google Scholar 

  • Jha AB, Sharma P (2019) Regulation of osmolytes syntheses in plants and improvement of abiotic stress tolerance: profiling and counteraction. In: Hasanuzzaman M, Nahar K, Fujita M, Oku H, Islam T (eds) Approaches for enhancing abiotic stress tolerance in plants, 1st edn. CRC Press Taylor and Francis, Boca Raton, pp 311–318

    Google Scholar 

  • Kalaiarasan E, Palvannan T (2015) Efficiency of carbohydrate additives on the stability of horseradish peroxidase (HRP): HRP-catalyzed removal of phenol and malachite green decolorization from wastewater. Clean (Weinh) 43:846–856

    CAS  Google Scholar 

  • Karam J, Nicell JA (1997) Potential applications of enzymes in waste treatment. J Chem Technol Biotechnol 69:141–153

    CAS  Google Scholar 

  • Kondyurin A, Nosworthy NJ, Bilek MMM (2015) Effect of low molecular weight additives on immobilization strength, activity, and conformation of protein immobilized on PVC and UHMWPE. Langmuir 27:6138–6148

    Google Scholar 

  • Kossowska D, Kwak K, Cho M (2018) Do osmolytes impact the structure and dynamics of myoglobin? Molecules 23:3189

    PubMed Central  Google Scholar 

  • Li Y, Zhang L, Fang S et al (2018) Variation of soil enzyme activity and microbial biomass in poplar plantations of different genotypes and stem spacings. J For Res 29:963–972

    CAS  Google Scholar 

  • Lineweaver H, Burk D (1934) The determination of enzyme dissociation constants. J Am Chem Soc 56:658–666

    CAS  Google Scholar 

  • Mahmoudi A, Nazari K, Moosavi-Movahedi AA (2003) Effect of Mn(II), Co(II), Ni(II) and Cu(II) on horseradish peroxidase: activation, inhibition and denaturation studies. Appl Biochemi Biotechnol 104:81–94

    CAS  Google Scholar 

  • Mishra S, Dubey RS (2006) Inhibition of ribonuclease and protease activities in arsenic exposed rice seedlings: role of proline as enzyme protectant. J Plant Physiol 163:927–936

    CAS  PubMed  Google Scholar 

  • Mohamad NR, Marzuki NHC, Buang NA, Huyop F, Wahab RA (2015) An overview of technologies for immobilization of enzymes and surface analysis techniques for immobilized enzymes. Biotechnol Biotechnol Equip 29:205–220

    CAS  PubMed  PubMed Central  Google Scholar 

  • Mojiri A, Zhou JL, Ohashi A, Ozaki N, Kindaichi T (2019) Comprehensive review of polycyclic aromatic hydrocarbons in water sources, their effects and treatments. Sci Total Environ 696:133971

    CAS  PubMed  Google Scholar 

  • Moreira PR, Almeida-Vara E, Malcata FX, Duarte JC (2007) Lignin transformation by a versatile peroxidase from a novel Bjerkandera sp. strain. Int Biodeterior Biodegr 59:234–238

    CAS  Google Scholar 

  • Na SY, Lee Y (2017) Elimination of trace organic contaminants during enhanced wastewater treatment with horseradish peroxidase/hydrogen peroxide (HRP/H2O2) catalytic process. Catal Today 282:86–94

    CAS  Google Scholar 

  • Ni Y, Li J, Huang Z, He K, Zhuang J, Yang W (2013) Improved activity of immobilized horseradish peroxidase on gold nanoparticles in the presence of bovine serum albumin. J Nanopart Res 15:2038

    Google Scholar 

  • Paleg LG, Stewart GR, Bradbeer JW (1984) Proline and glycine betaine influence protein solvation. Plant Physiol 75(4):974–978

    CAS  PubMed  PubMed Central  Google Scholar 

  • Samuel D, Kumar TK, Ganesh G, Jayaraman G, Yang PW, Chang MM, Trivedi VD, Wang SL, Hwang KC, Chang DK, Yu C (2000) Proline inhibits aggregation during protein refolding. Protein Sci 9:344–352

    CAS  PubMed  PubMed Central  Google Scholar 

  • Schobert B, Tschesche H (1978) Unusual solution properties of proline and its interaction with proteins. BBA-Gen Subj 541(2):270–277

    CAS  Google Scholar 

  • Sharma P, Dubey RS (2004) Ascorbate peroxidase from rice seedlings; properties of enzyme isoforms, effects of stress and protective roles of osmolytes. Plant Sci 167:541–550

    CAS  Google Scholar 

  • Sharma P, Dubey RS (2005) Modulation of nitrate reductase activity in rice seedlings under aluminium toxicity and water stress: role of osmolytes as enzyme protectant. J Plant Physiol 162:854–864

    CAS  PubMed  Google Scholar 

  • Sharma SS, Schat H, Voojis R (1998) In vitro alleviation of heavy metal-induced enzyme inhibition by proline. Phytochemistry 49:1531–1535

    CAS  PubMed  Google Scholar 

  • Shi X, Dalal S, Kasprzak KS (1992) Generation of free radicals from lipid hydroperoxides by Ni2+ in the presence of oligopeptides. Arch Biochem Biophys 299:154–162

    CAS  PubMed  Google Scholar 

  • Singh S, Jha AB, Misra AN, Sharma P (2017) Amorphophallus paeoniifolius corm: a potential source of peroxidase for wide applications. Int J Food Prop 20:2658–2664

    CAS  Google Scholar 

  • Unuofin JO, Okoh AI, Nwodo UU (2019) Aptitude of oxidative enzymes for treatment of wastewater pollutants: a laccase perspective. Molecules 24:2064

    CAS  PubMed Central  Google Scholar 

  • Veitch NC (2004) Horseradish peroxidase: a modern view of a classic enzyme. Phytochemistry 65:249–259

    CAS  PubMed  Google Scholar 

  • Vineh MB, Saboury AA, Poostchi AA, Chi AA, Rashidi AM, Parivar K (2018) Stability and activity improvement of horseradish peroxidase by covalent immobilization on functionalized reduced graphene oxide and biodegradation of high phenol concentration. Int J Biol Macromo l106:1314–1322

    Google Scholar 

  • Xie G, Timasheff SN (1997) The thermodynamic mechanism of protein stabilization by trehalose. Biophys Chem 64:25–43

    CAS  PubMed  Google Scholar 

  • Zheng G, Liu S, Zha J, Zhang P, Xu X, Chen Y, Jiang S (2019) Protecting enzymatic activity via zwitterionic nanocapsulation for the removal of phenol compound from wastewater. Langmuir 35:1858–1863

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

PS is thankful to UGC-Start-up Grant no. F.4-5(107-FRP)/2014(BSR) and DST-SERB Project no. ECR/2016/000888 for financial support. DBT Builder project no. BT/PR-9028/INF/22/193/2013 is greatly acknowledged. RS is thankful for CUJ University Fellowship during the period of this work.

Author information

Authors and Affiliations

  1. Department of Life Sciences, Central University of Jharkhand, Brambe, Ranchi, 835205, India

    Rajani Singh, Amarendra Narayan Misra & Pallavi Sharma

  2. Crop Development Centre, Department of Plant Sciences, University of Saskatchewan, Saskatoon, SK, S7N5A8, Canada

    Ambuj Bhushan Jha

  3. Khallikote University, Berhampur, Odisha, 761008, India

    Amarendra Narayan Misra

Authors
  1. Rajani Singh
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ambuj Bhushan Jha
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Amarendra Narayan Misra
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Pallavi Sharma
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Pallavi Sharma.

Ethics declarations

Conflict of interest

There is no conflict of interest related to this study.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Singh, R., Jha, A.B., Misra, A.N. et al. Entrapment of enzyme in the presence of proline: effective approach to enhance activity and stability of horseradish peroxidase. 3 Biotech 10, 155 (2020). https://doi.org/10.1007/s13205-020-2140-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s13205-020-2140-7

Keywords

Search

Navigation