Ultra-Thin 2D CuO Nanosheet for HRP Immobilization Supported by Encapsulation in a Polymer Matrix: Characterization and Dye Degradation

Abstract

Herein, we report the immobilization of horseradish peroxidase (HRP) on a novel two-dimensional copper oxide nanosheet (CuONS) and supported by encapsulation in poly (methyl methacrylate) (PMMA). The morphological/chemical properties of the immobilized HRP on CuONS-PMMA were investigated and characterized by scanning electron microscopy, and Fourier transforms infrared spectroscopy. After optimizing the immobilization conditions, recovered HRP activity was 72.8% using 1% CuONS–PMMA. Reusing the immobilized enzyme for ten cycles indicated that 52% of its initial activity was retained. Moreover, the immobilization of HRP using this method led to a shift in its optimum pH from 7 to broad optimum 7–7.5 and optimum temperature from 40 °C to broad optimum 40–50 °C. Km values of free and immobilized HRP were 30 and 46.7 mM for guaiacol and 5.53 and 8.55 mM for H2O2, respectively. The immobilized HRP enzyme was more stable towards metal ions. The immobilized HRP was used for crystal violet, methyl green, and malachite green dyes degradation. The immobilized HRP showed significant improvement in the capacity for dye decolorization relative to free enzyme. Therefore, based on all the characteristics mentioned above, it may be suggested that the use of this technique for HRP immobilization appears to be promising for industrial applications.

Graphic Abstract

A schematic illustration of the immobilization process of HRP on CuONS/PMMA.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Scheme 1
Fig. 8

References

  1. 1.

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

    CAS  Google Scholar 

  2. 2.

    Bilal M, Rasheed T, Iqbal HM, Hu H, Wang W, Zhang X (2018) Horseradish peroxidase immobilization by copolymerization into cross-linked polyacrylamide gel and its dye degradation and detoxification potential. Int J Boil Macromol 113:983–990

    CAS  Google Scholar 

  3. 3.

    Bilal M, Asgher M, Cheng H, Yan Y, Iqbal HM (2019) Multi-point enzyme immobilization, surface chemistry, and novel platforms: a paradigm shift in biocatalyst design. Crit Rev Biotechnol 39:202–219

    CAS  PubMed  Google Scholar 

  4. 4.

    Jun LY, Mubarak N, Yon LS, Bing CH, Khalid M, Jagadish P, Abdullah E (2019a) Immobilization of peroxidase on functionalized MWCNTs-buckypaper/polyvinyl alcohol nanocomposite membrane. Sci Rep 9:2215

    PubMed  PubMed Central  Google Scholar 

  5. 5.

    Jun LY, Yon LS, Mubarak N, Bing CH, Pan S, Danquah MK, Abdullah E, Khalid M (2019b) An overview of immobilized enzyme technologies for dye, phaenolic removal from wastewater. J Environ Chem Eng 7:102961

    CAS  Google Scholar 

  6. 6.

    Bilal M, Iqbal HM (2019a) Chemical, physical, and biological coordination: an interplay between materials and enzymes as potential platforms for immobilization. Coordin Chem Rev 388:1–23

    CAS  Google Scholar 

  7. 7.

    Eş I, Vieira JDG, Amaral AC (2015) Principles, techniques, and applications of biocatalyst immobilization for industrial application. Appl Microb Biotechnol 99:2065–2082

    Google Scholar 

  8. 8.

    Aldhahri MM, Almulaiky YQ, El-Shishtawy RM, Al-Shawafi W, Alngadh A, Maghrabi R (2018) Facile immobilization of enzyme via co-electrospinning: a simple method for enhancing enzyme reusability and monitoring an activity-based organic semiconductor. ACS Omega 3:6346–6350

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Huang WC, Wang W, Xue C, Mao X (2018) Effective enzyme immobilization onto a magnetic chitin nanofiber composite, ACS sustain. Chem Eng 6:8118–8124

    CAS  Google Scholar 

  10. 10.

    Jesionowski T, Zdarta J, Krajewska B (2014) Enzyme immobilization by adsorption: a review. Adsorption. 20:801–821

    CAS  Google Scholar 

  11. 11.

    Song J, He W, Shen H, Zhou Z, Li M, Su P, Yang Y (2019) Self-assembly of a magnetic DNA hydrogel as a new biomaterial for enzyme encapsulation with enhanced activity and stability. Chem Comm 55:2449–2452

    CAS  PubMed  Google Scholar 

  12. 12.

    Xu R, Zhou Q, Li F, Zhang B (2013) Laccase immobilization on chitosan/poly (vinyl alcohol) composite nanofibrous membranes for 2, 4-dichlorophenol removal. Chem Eng J 222:321–329

    CAS  Google Scholar 

  13. 13.

    Wong JKH, Tan HK, Lau SY, Yap PS, Danquah MK (2019) Potential and challenges of enzyme incorporated nanotechnology in dye wastewater treatment: a review. J Environ Chem Eng 7:103261

    CAS  Google Scholar 

  14. 14.

    Hwang ET, Gu MB (2013) Enzyme stabilization by nano/microsized hybrid materials. Eng Life Sci 13:49–61

    CAS  Google Scholar 

  15. 15.

    Brena B, González-Pombo P, Batista-Viera F (2013) Immobilization of enzymes: a literature survey. In: Immobilization of enzymes and cells. Springer, Ney York, pp 15–31

    Google Scholar 

  16. 16.

    Chang TMS (1977) Encapsulation of enzymes, cell contents, cells, vaccines, antigens, antiserum, cofactors, hormones, and proteins. In: Biomedical applications of immobilized enzymes and proteins. Springer, Boston, MA, pp 69–90

    Google Scholar 

  17. 17.

    Chang MY, Juang RS (2007) Use of chitosan–clay composite as immobilization support for improved activity and stability of β-glucosidase. Biochem Eng J 35:93–98

    CAS  Google Scholar 

  18. 18.

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

    Google Scholar 

  19. 19.

    Bilal M, Iqbal HM (2019b) Naturally-derived biopolymers: potential platforms for enzyme immobilization. J Bio Macromol 1:462–482

    Google Scholar 

  20. 20.

    Gao X, Ding Y, Sheng Y, Hu M, Zhai Q, Li S, JiangY CY (2019) Enzyme immobilization in MOF-derived porous NiO with hierarchical structure: an efficient and stable enzymatic reactor. Chem Cat Chem 11:2828–2836

    CAS  Google Scholar 

  21. 21.

    Ge J, Lei J, Zare RN (2012) Protein–inorganic hybrid nanoflowers. Nature Nanotechnol 7:428

    CAS  Google Scholar 

  22. 22.

    Mohamed SA, Al-Harbi MH, Almulaiky YQ, Ibrahim IH, Salah HA, El-Badry MO, Abdel-Aty AM, Fahmy AS, El-Shishtawy RM (2018) Immobilization of Trichoderma harzianum α-amylase on PPyAgNp/Fe3O4-nanocomposite: chemical and physical properties. Artif Cells Nanomed Biotechnol 46:201–206

    CAS  PubMed  Google Scholar 

  23. 23.

    Zhang Y, Yue Q, Zagho MM, Zhang J, Elzatahry AA, Jiang Y, Deng Y (2019) Core–Shell magnetic Mesoporous silica microspheres with large Mesopores for enzyme immobilization in biocatalysis. ACS Appl Mater Inter 11:10356–10363

    CAS  Google Scholar 

  24. 24.

    Liu Z, Jiang Y, Liu X, Zeng G, Shao B, Liu Y, He X (2019) Silver chromate modified sulfur doped graphitic carbon nitride microrod composites with enhanced visible-light photoactivity towards organic pollutants degradation. Compos Part B Eng 173:106918

    CAS  Google Scholar 

  25. 25.

    Shao B, Liu X, Liu Z, Zeng G, Zhang W, Liang Q, Luo S (2019) Synthesis and characterization of 2D/0D g-C3N4/CdS-nitrogen doped hollow carbon spheres (NHCs) composites with enhanced visible light photodegradation activity for antibiotic. Chem Eng J:374, 479–493

  26. 26.

    Shao B, Liu Z, Zeng G, Wang H, Liang Q, He Q, Song B (2020) Two-dimensional transition metal carbide and nitride (MXene) derived quantum dots (QDs): synthesis, properties, applications and prospects. J Mater Chem A 8:7508–7535

    CAS  Google Scholar 

  27. 27.

    Siddiqui MTH, Nizamuddin S, Baloch HA, Mubarak NM, Al-Ali M, Mazari SA, Griffin G (2019) Fabrication of advance magnetic carbon nano-materials and their potential applications: a review. J Environ Chem Eng 7:102812

    CAS  Google Scholar 

  28. 28.

    Yang J, Zeng Z, Kang J, Betzler S, Czarnik C, Zhang X, Ophus C, Yu C, Bustillo K, Pan M (2019) Formation of two-dimensional transition metal oxide nanosheets with nanoparticles as intermediates. Nat Mater 18:970–976

    Google Scholar 

  29. 29.

    Jun LY, Karri RR, Yon LS, Mubarak NM, Bing CH, Mohammad K, Abdullah EC (2020) Modeling and optimization by particle swarm embedded neural network for adsorption of methylene blue by jicama peroxidase immobilized on buckypaper/polyvinyl alcohol membrane. Environ Res 183:109158

    CAS  PubMed  Google Scholar 

  30. 30.

    Wen W, Song Y, Yan X, Zhu C, Du D, Wang S, Asiri AM, Lin Y (2018) Recent advances in emerging 2D nanomaterials for biosensing and bioimaging applications. Mater Today 21:164–177

    CAS  Google Scholar 

  31. 31.

    Mei S, Shi J, Zhang S, Wang Y, Wu Y, Jiang Z, Wu H (2019) Nanoporous phyllosilicate assemblies for enzyme immobilization. ACS Appl Bio Mater 2:777–786

    CAS  Google Scholar 

  32. 32.

    Pawar SM, Pawar BS, Hou B, Kim J, Ahmed ATA, Chavan HS, Jo Y, Cho S, Inamdar AI, Gunjakar JL (2017) Self-assembled two-dimensional copper oxide nanosheet bundles as an efficient oxygen evolution reaction (OER) electrocatalyst for water splitting applications. J Mater Chem A 5:12747–12751

    CAS  Google Scholar 

  33. 33.

    Salah N, Alshahrie A, Abdel-wahab MS, Alharbi ND, Khan ZH (2017) Carbon nanotubes of oil fly ash integrated with ultrathin CuO nanosheets as effective lubricant additives. Diam. Relat Mater 78:97–104

    CAS  Google Scholar 

  34. 34.

    Salah N, Baghdadi N, Alshahrie A, Saeed A, Ansari A, Memic A, Koumoto K (2019) Nanocomposites of CuO/SWCNT: promising thermoelectric materials for mid-temperature thermoelectric generators. J Eur Ceram Soc 39:3307–3314

    CAS  Google Scholar 

  35. 35.

    Shinde S, Dhaygude H, Kim DY, Ghodake G, Bhagwat P, Dandge P, Fulari V (2016) Improved synthesis of copper oxide nanosheets and its application in development of supercapacitor and antimicrobial agents. J Ind Eng Chem 36:116–120

    CAS  Google Scholar 

  36. 36.

    Shahmiri M, Ibrahim NA, Yunus WMZW, Shameli K, Zainuddin N, Jahangirian H (2013) Synthesis and characterization of CuO nanosheets in polyvinylpyrrolidone by quick precipitation method. Adv Sci EnginMed 5:193–197

    CAS  Google Scholar 

  37. 37.

    Yuan WZ, Shen XY, Zhao H, Lam JW, Tang L, Lu P, Sun JZ (2010) Crystallization-induced phosphorescence of pure organic luminogens at room temperature. J Phys Chem C 114:6090–6099

    CAS  Google Scholar 

  38. 38.

    Kvashnin DG, Kvashnin AG, Kano E, Hashimoto A, Takeguchi M, Naramoto H, Sorokin PB (2019) Two-dimensional CuO inside the supportive bilayer graphene matrix. J Phys Chem C 123:17459–17465

    CAS  Google Scholar 

  39. 39.

    Umar A, Rahman MM, Al-Hajry A, Hahn YB (2009) Enzymatic glucose biosensor based on flower-shaped copper oxide nanostructures composed of thin nanosheets. Electrochem 11:278–281

    CAS  Google Scholar 

  40. 40.

    Bilal M, Rasheed T, Iqbal HM, Hu H, Wang W, Zhang X (2017) Novel characteristics of horseradish peroxidase immobilized onto the polyvinyl alcohol-alginate beads and its methyl orange degradation potential. Int J Boil Macromol 105:328–335

    CAS  Google Scholar 

  41. 41.

    Li J, Chen X, Xu D, Pan K (2019a) Immobilization of horseradish peroxidase on electrospun magnetic nanofibers for phenol removal. Ecotox Environ Safe 170:716–721

    CAS  Google Scholar 

  42. 42.

    Li Y, Sun L, Zhao Q (2019b) Aptamer-structure switch coupled with horseradish peroxidase Labeling on a microplate for the sensitive detection of small molecules. Analyt Chem 91:2615–2619

    CAS  Google Scholar 

  43. 43.

    Mejri F, Karmali A, Jaoued N, Casabianca H, Hosni K (2019) Purification and partial characterization of peroxidases from three food waste by-products: broad bean pods, pea pods, and artichoke stems. Appl Biochem Biotechnol 189:576–588

    CAS  PubMed  Google Scholar 

  44. 44.

    Zhang Z, Lai J, Wu K, Huang X, Guo S, Zhang L, Liu J (2018) Peroxidase-catalyzed chemiluminescence system and its application in immunoassay. Talanta 180:260–270

    CAS  PubMed  Google Scholar 

  45. 45.

    Rodríguez-López JN, Lowe DJ, Hernández-Ruiz J, Hiner AN, García-Cánovas F, Thorneley RN (2001) Mechanism of reaction of hydrogen peroxide with horseradish peroxidase: identification of intermediates in the catalytic cycle. J Am Chem Soc 123:11838–11847

    PubMed  Google Scholar 

  46. 46.

    Nahas MN, Jilani A, Salah N (2016) Microwave synthesis of ultrathin, non-agglomerated CuO nanosheets and their evaluation as nanofillers for polymer nanocomposites. J Alloys Compd 680:350–358

    CAS  Google Scholar 

  47. 47.

    Yuan ZY, Jiang TJ (2003) Horseradish peroxidase. In: Whitaker JR, Voragen A, Wong DWS (eds) Handbook of food enzymology. Marcel Dekker Inc, New York, pp 403–411

    Google Scholar 

  48. 48.

    Parshetti GK, Parshetti S, Kalyani DC, Doong RA, Govindwar SP (2012) Industrial dye decolorizing lignin peroxidase from Kocuria rosea MTCC 1532. Ann Micro Biol 62:217–223

    CAS  Google Scholar 

  49. 49.

    Karim Z, Adnan R, Ansari MS (2012a) Low concentration of silver nanoparticles not only enhances the activity of horseradish peroxidase but alter the structure also. PLoS One 7:e41422

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Karim Z, Adnan R, Husain Q (2012b) A β-cyclodextrin–chitosan complex as the immobilization matrix for horseradish peroxidase and its application for the removal of azo dyes from textile effluent. Int Biodeterior Biodegradation 72:10–17

    CAS  Google Scholar 

  51. 51.

    Mallakpour S, Jarahiyan A (2017) Surface treatment of copper (II) oxide nanoparticles using citric acid and ascorbic acid as biocompatible molecules and their utilization for the preparation of poly (vinyl chloride) novel nanocomposite films. J Thermoplast Compos Mater 30:1267–1284

    CAS  Google Scholar 

  52. 52.

    Alshawafi WM, Aldhahri M, Almulaiky YQ, Salah N, Moselhy SS, Ibrahim IH, El-Shishtawy RM, Mohamed SA (2018) Immobilization of horseradish peroxidase on PMMA nanofibers incorporated with nanodiamond. Artif Cell Nanomed B 46:S973–S981

    CAS  Google Scholar 

  53. 53.

    Hussein MA, El-Shishtawy RM, Abu-Zied BM, Asiri AM (2016) The impact of cross-linking degree on the thermal and texture behavior of poly (methyl methacrylate). J Therm Anal Calorim 124:709–717

    CAS  Google Scholar 

  54. 54.

    Mohamed SA, Al-Malki AL, Kumosani TA, El-Shishtawy RM (2013a) Horseradish peroxidase and chitosan: activation, immobilization and comparative results. Int J Bio Macromol 60:295–300

    CAS  Google Scholar 

  55. 55.

    Mohamed SA, Darwish AA, El-Shishtawy RM (2013b) Immobilization of horseradish peroxidase on activated wool. Process Biochem 48:649–655

    CAS  Google Scholar 

  56. 56.

    Mazlan SZ, Hanifah SA (2017) Effects of temperature and pH on immobilized laccase activity in conjugated methacrylate-acrylate microspheres. Int J Polym Sci 2017:1–8

    Google Scholar 

  57. 57.

    Shaked E, Shani Y, Zilberman M, Scheinowitz M (2015) Poly (methyl methacrylate) particles for local drug delivery using shock wave lithotripsy: I n vitro proof of concept experiment. J Biomed Mater Res B Appl Biomater 10:1228–1237

    Google Scholar 

  58. 58.

    Abdullah J, Ahmad M, Heng LY, Karuppiah N, Sidek H (2007) An optical biosensor based on immobilization of laccase and MBTH in stacked films for the detection of catechol. Sensors. 7:2238–2250

    CAS  PubMed  Google Scholar 

  59. 59.

    Querol E, Perez-Pons JA, Mozo-Villarias A (1996) Analysis of protein conformational characteristics related to thermostability. Protein Eng Des Sel 9:265–271

    CAS  Google Scholar 

  60. 60.

    Mohamed SA, Al-Harbi MH, Almulaiky YQ, Ibrahim IH, El-Shishtawy RM (2017) Immobilization of horseradish peroxidase on Fe3O4 magnetic nanoparticles. Electron J Biotechnol 27:84–90

    CAS  Google Scholar 

  61. 61.

    Pandey VP, Rani J, Jaiswal N, Singh S, Awasthi M, Shasany AK, Tiwari S, Dwivedi UN (2017) Chitosan immobilized novel peroxidase from Azadirachta indica: characterization and application. Int J Bio Macromol 104:1713–1720

    CAS  Google Scholar 

  62. 62.

    Ashraf, H, Husain, Q. (2010). Stabilization of DEAE cellulose adsorbed and glutaraldehyde crosslinkedwhite radish (Raphanus sativus) peroxidase. J Sci Ind Res India 69:613–620

    CAS  Google Scholar 

  63. 63.

    Tanford C (1970) Theoretical models for the mechanism ofdenaturation. Adv Protein Chem 24:2–97

    Google Scholar 

  64. 64.

    Pandey VP, Dwivedi UN (2011) Purification and characterization of peroxidase from Leucaena leucocephala, a tree legume. J Mol Catal B 68:168–173

    CAS  Google Scholar 

  65. 65.

    Almulaiky YQ, Al-Harbi SA (2019) A novel peroxidase from Arabian balsam (Commiphora gileadensis) stems: its purification, characterization and immobilization on a carboxymethylcellulose/Fe3O4 magnetic hybrid material. Int J Boil Macromol 133:767–774

    CAS  Google Scholar 

  66. 66.

    Chanwun T, Muhamad N, Chirapongsatonkul N, Churngchow N (2013) Hevea brasiliensis cell suspension peroxidase: purification, characterization and application for dye decolorization. AMB Express 3:14

    PubMed  PubMed Central  Google Scholar 

  67. 67.

    Al-Angari YM, Ewais HA, El-Shishtawy RM (2019) Kinetics and mechanism of the oxidative decolorization of direct violet 31 in the presence of peroxodisulfate-silver(I) as a redox system. Transit Met Chem 44:57–64

    CAS  Google Scholar 

  68. 68.

    Chen SX, Schopfer P (1999) Hydroxyl-radical production in physiological reactions a novel function of peroxidase. Eur J Biochem 260:726–735

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We would like to acknowledge the center of nanotechnology, king Abdulaziz University and university of Jeddah for providing the support for characterization.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Yaaser Q. Almulaiky.

Ethics declarations

Conflict of interest

Authors declare that they have no conflict of interest.

Additional information

Publisher's Note

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

Electronic supplementary material

Below is the link to the electronic supplementary material.

Electronic supplementary material 1 (DOCX 1527 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Aldhahri, M., Almulaiky, Y.Q., El-Shishtawy, R.M. et al. Ultra-Thin 2D CuO Nanosheet for HRP Immobilization Supported by Encapsulation in a Polymer Matrix: Characterization and Dye Degradation. Catal Lett (2020). https://doi.org/10.1007/s10562-020-03289-7

Download citation

Keywords

  • Immobilization
  • Enzymes
  • 2D nanomaterials
  • Copper oxide nanosheets
  • Dye decolorization