Co-immobilization of multiple enzymes onto surface-functionalized magnetic nanoparticle for the simultaneous hydrolysis of multiple substrates containing industrial wastes

  • S. Hepziba Suganthi
  • K. V. Swathi
  • Raagini Biswas
  • Sneha Basker
  • K. RamaniEmail author
Original Article


The present study focused on the co-immobilization of multi-enzymes onto magnetic nanoparticles (MNPs) and surface-functionalized MNPs for the hydrolysis of fish processing solid waste (FPSW) containing complex molecules. The MNPs were synthesized and surface-functionalized by chemical (cMNP and SF-cMNP) and biological method [biologically synthesized iron oxide magnetic nanoparticle (bIOMNP)] and was characterized by various instrumental analyses. Lipase and protease produced from Streptomyces thermolineatus were co-immobilized onto the synthesized bare and surface-functionalized MNPs. At the optimum conditions, the lipase and protease loading capacity of cMNP were found as 1000 and 783 U/g, respectively, whereas the lipase and protease loading capacity of SF-cMNP were 2920 and 2350 U/g, respectively, and bIOMNP was 2800 and 2196 U/g, respectively. The thermal, pH and storage stabilities of the co-immobilized enzymes were greatly enhanced compared to the free enzymes and the Vmax and Km of the free and co-immobilized enzymes were also determined. The free and the co-immobilized enzymes were used for the hydrolysis of lipids and proteins in FPSW. The percentage hydrolysis of lipids and proteins in FPSW was 52.8 and 61.8%, respectively, for LP-cMNP, 76.2 and 84.1%, respectively, for LP-SF-cMNP and 73.9 and 82.1%, respectively, for LP-bIOMNP.


Fish processing solid waste Iron oxide magnetic nanoparticles Surface functionalization Enzyme co-immobilization Multi-enzyme catalysis 



The authors are grateful to the Department of Biotechnology, Nanotechnology research centre, Department of Physics & Nanotechnology, SRM Institute of Science and Technology and SAIF, IIT-Madras for extending the analytical facilities.

Compliance with ethical standards

Conflict of interest



  1. Abdollahi M, Zeinali S, Nasirimoghaddam S, Sabbaghi S (2014) Effective removal of As (III) from drinking water samples by chitosan-coated magnetic nanoparticles. Desalin Water Treat 56:2092–2104CrossRefGoogle Scholar
  2. Adeogun AI, Kareeem SO, Adebayo OS, Balogun SA (2017) Comparative adsorption of amylase, protease and lipase on ZnFe2O4: kinetics, isothermal and thermodynamics studies. 3 Biotech 7:198CrossRefGoogle Scholar
  3. Asmat S, Husain Q, Azam A (2017) Lipase immobilization on facile synthesized polyaniline-coated silver-functionalized graphene oxide nanocomposites as novel biocatalysts: stability and activity insights. RSC Adv 7:5019–5029CrossRefGoogle Scholar
  4. Badruddoza AZM, Tay ASH, Tan PY et al (2011) Carboxymethy- β cyclodextrin conjugated magnetic nanoparticles as nano-adsorbents for removal of copper ions: synthesis and adsorption studies. J Hazard Mater 185:1177–1186CrossRefGoogle Scholar
  5. 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 72:248–254CrossRefGoogle Scholar
  6. Cho EJ, Jung S, Kim HJ et al (2012) Co-immobilization of three cellulases on Au-doped magnetic silicananoparticles for the degradation of cellulose. Chem Commun 48:886–888CrossRefGoogle Scholar
  7. Chong S, Zhang G, Tian H, He Z (2016) Rapid degradation of dyes in water by magnetic Fe0/Fe3O4/graphene composites. J Environ Sci 44:148–157CrossRefGoogle Scholar
  8. Demir D, Gures D, Tecim T et al (2018) Magnetic nanoparticle-loaded electrospun poly(ε-caprolactone) nanofibers for drug delivery applications. Appl Nanosci 8:1461–1469CrossRefGoogle Scholar
  9. Dutta N, Biswas S, Saha MK (2016) Biophysical characterization and activity analysis of nano-magnesium supplemented cellulase obtained from a psychrobacterium following graphene oxide immobilization. Enzyme Microb Technol. Google Scholar
  10. Gao Z, Yi Y, Zhao J et al (2018) Co-immobilization of laccase and TEMPO onto amino-functionalized magnetic nanoparticles and its application in acid fuchsin decolorization. Bioresour Bioprocess 5:27CrossRefGoogle Scholar
  11. Homaei AA, Sariri R, Vianello F, Stevanato R (2013) Enzyme immobilization: an update. J Chem Biol 6:185–205CrossRefGoogle Scholar
  12. Hu B, Pan J, Yu HL et al (2009) Immobilization of Serratia marcescens lipase onto amino-functionalized magnetic nanoparticles for repeated use in enzymatic synthesis of Diltiazem intermediate. Process Biochem 44:1019–1024CrossRefGoogle Scholar
  13. Hwang ET, Gu MB (2013) Enzyme stabilization by nano/microsized hybrid materials. Eng Life Sci 13:49–61CrossRefGoogle Scholar
  14. Jayasinghe P, Hawboldt K (2013) Biofuels from fish processing plant effluents waste characterization and oil extraction and quality. Sustain Energy Technol Assessments 4:36–44CrossRefGoogle Scholar
  15. Jin X, Li S, Long N, Zhang R (2018) A robust and stable nano-biocatalyst by co-immobilization of chloroperoxidase. J Chem Technol Biotechnol 93:489–497CrossRefGoogle Scholar
  16. Khanjanzadeh H, Behrooz R, Bahramifar N et al (2018) Surface chemical functionalization of cellulose nanocrystals by 3-aminopropyltriethoxysilane. Int J Biol Macromol 106:1288–1296CrossRefGoogle Scholar
  17. Knight JA, Anderson S, Rawle JM (1972) Chemical basis of the sulfo-phospho-vanillin reaction for estimating total serum lipids. Clin Chem 18:199–202Google Scholar
  18. Kumar GA, Venkatesan R, Kirubagaran R et al (2008) Effects of nonionic surfactant on hydrolysis and fermentation of protein rich tannery solid waste. Biodegradation 19:739–748CrossRefGoogle Scholar
  19. Li C, Jiang S, Zhao X, Liang H (2017) Co-immobilization of enzymes and magnetic nanoparticles by metal-nucleotide hydrogel nanofibers for improving stability and recycling. Molecules 22:179–190CrossRefGoogle Scholar
  20. Liang H, Jiang S, Yuan Q et al (2016) Co-immobilization of multiple enzymes by metal coordinated nucleotide hydrogel nanofibers: improved stability and an enzyme cascade for glucose detection. Nanoscale 8:6071–6078CrossRefGoogle Scholar
  21. Liu Y, Chen T, Wu C et al (2014) Facile surface functionalization of hydrophobic magnetic nanoparticles. J Am Chem Soc 136:12552–12555CrossRefGoogle Scholar
  22. Motevalizadeh SF, Khoobi M, Sadighi A et al (2015) Lipase immobilization onto polyethylenimine coated magnetic nanoparticles assisted by divalent metal chelated ions. J Mol Catal B Enzym 120:75–83CrossRefGoogle Scholar
  23. Nasrollahzadeh M, Sajadi SM (2016) Green synthesis of Pd nanoparticles mediated by Euphorbia thymifolia L. leaf extract: catalytic activity for cyanation of aryl iodides under ligand-free conditions. J Colloid Interface Sci 469:191–195CrossRefGoogle Scholar
  24. Pashangeh K, Akhond M, Heidari KHR, Absalan G (2017) Biochemical characterization and stability assessment of Rhizopus oryzae lipase covalently immobilized on amino-functionalized magnetic nanoparticles. Int J Biol Macromol 105:300–307CrossRefGoogle Scholar
  25. Petrovicova T, Markosova K, Hegyi Z et al (2018) Co-immobilization of ketoreductase and glucose dehydrogenase. Catalysts 8:168CrossRefGoogle Scholar
  26. Ramani K, Boopathy R, Mandal AB, Sekaran G (2011) Preparation of acidic lipase immobilized surface-modified mesoporous activated carbon catalyst and thereof for the hydrolysis of lipids. Catal Commun 14:82–88CrossRefGoogle Scholar
  27. Sahu A, Badhe PS, Adivarekar R et al (2016) Synthesis of glycinamides using protease immobilized magnetic nanoparticles. Biotechnol Rep 12:13–25CrossRefGoogle Scholar
  28. Saranya P, Ramani K, Sekaran G (2014) Biocatalytic approach on the treatment of edible oil re fi nery wastewater. RSC Adv 4:10680–10692CrossRefGoogle Scholar
  29. Shah ST, Yehya A, Saad O et al (2017) Surface functionalization of iron oxide nanoparticles with gallic acid as potential antioxidant and antimicrobial agents. Nanomaterials 7:306–323CrossRefGoogle Scholar
  30. Shi Y, Liu W, Tao Q-L et al (2016) Immobilization of lipase by adsorption onto magnetic nanoparticles in organic solvents. J Nanosci Nanotechnol 16:601–607CrossRefGoogle Scholar
  31. Soares PIP, Machado D, Laia C et al (2016) Thermal and magnetic properties of chitosan-iron oxide nanoparticles. Carbohydr Polym 149:382–390CrossRefGoogle Scholar
  32. Songvorawit N, Tuitemwong K, Tuitemwong P (2011) Single step synthesis of amino-functionalized magnetic nanoparticles with polyol technique at low temperature. ISRN Nanotechnol 2011:1–6CrossRefGoogle Scholar
  33. Suganthi HS, Kandasamy R (2017) A novel single step synthesis and surface functionalization of iron oxide magnetic nanoparticles and thereof for the copper removal from pigment industry effluent. Sep Purif Technol 188:458–467CrossRefGoogle Scholar
  34. Suganthi SH, Ramani K (2016) Microbial assisted industrially important multiple enzymes from fish processing waste: purification, characterization and application for the simultaneous hydrolysis of lipid and protein molecules. RSC Adv 6:93602–93620CrossRefGoogle Scholar
  35. Wu W, He Q, Jiang C (2008) Magnetic iron oxide nanoparticles: synthesis and surface functionalization strategies. Nanoscale Res Lett 3:397–415CrossRefGoogle Scholar
  36. Yamaura M, Camilo RL, Sampaio LC et al (2004) Preparation and characterization of (3-aminopropyl) triethoxysilane-coated magnetite nanoparticles. J Magn Magn Mater 279:210–217CrossRefGoogle Scholar
  37. Yang H-W, Hua M-Y, Liu H-L et al (2012) Potential of magnetic nanoparticles for targeted drug delivery. Nanotechnol Sci Appl 5:73–86Google Scholar
  38. Yazid NA, Barrena R, Sanchez A (2016) The immobilisation of proteases produced by SSF onto functionalized magnetic nanoparticles: application in the hydrolysis of different protein sources. J Mol Catal B Enzym 133:S230–S242CrossRefGoogle Scholar
  39. Yemm EW, Cocking EC, Ricketts RE (1955) The determination of amino-acids with ninhydrin. Analyst 80:209–214CrossRefGoogle Scholar

Copyright information

© King Abdulaziz City for Science and Technology 2019

Authors and Affiliations

  • S. Hepziba Suganthi
    • 1
  • K. V. Swathi
    • 1
  • Raagini Biswas
    • 1
  • Sneha Basker
    • 1
  • K. Ramani
    • 1
    Email author
  1. 1.Biomolecules and Biocatalysis Laboratory, Department of BiotechnologySRM Institute of Science and TechnologyKattankulathurIndia

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