Co-immobilization of α-amylase and Maltogenic Amylase by Nanomagnetic Combi-Cross-linked Enzyme Aggregates for High Maltose syrup Production From Corn Starch

Document Type : Research Article


1 M.S. Student, Department of Agriculture Research, Iranian Research Organization for Science and Technology (IROST)

2 Assistant Professor, Department of Chemical Technology , Iranian Research Organization for Science and Technology (IROST)


In this study Co-immobilization of α-amylase and maltogenic amylase by using of lysine functionalized magnetic nanoparticles cross-linked enzyme aggregates method was carried out for high maltose syrup production from corn starch. Nanomanetite (Fe3O4) was prepared by co-precipitation method and then, surface functionalization by lysine amino acid was achieved. Enzyme aggregation was performed by acetonitrile, acetone, tert-butanol, isopropanol, ethanol, and saturated ammonium sulfate that tert-butanol implied high enzyme activity for immobilization. Enzyme ratio of 1:9 (α-amylase: maltogenic amylase) optimum pH 6 and optimum temperature 65º C, 2 mM glutaraldehyde concentration, and enzyme to lysine ratio of 1:0.75 were the best conditions for enzyme immobilization. DLS results indicated that, mean particle size of nanomagnetites was in the range of 81.9-88.9 nm with PDI= 0.242 and zeta potential of -21 and mean particle size of immobilized enzyme was in the range of 99.60-110.5 nm with PDI= 0.088 and zeta potential of -32 respectively. The appearance of new NH2 and NH spectra in FTIR spectroscopy confirmed the enzyme''''s immobilization on magnetic nanoparticles. Moreover, immobilized enzyme preserved 80.36% of its activity after 10 consecutive cycles. Comparative study on kinetic parameters of immobilized enzyme versus free one revealed that, Vmax had not any significant change while, Km was reduced 1.5 time. The enzyme half-life of immobilized enzyme increased 2.5 times at 95º C compared to free one. Enzyme loading of immobilized enzyme on magnetic nanoparticles were determined 82%.

Graphical Abstract

Co-immobilization of α-amylase  and Maltogenic Amylase by Nanomagnetic Combi-Cross-linked Enzyme Aggregates for High Maltose syrup Production From Corn Starch


  • A novel nanomagnetic combi-CLEAs method which converts starch into maltose.
  • Efficient starch conversion to maltose obtaining by nano co-immobilized amylases.
  • Reusable NM-combi-CLEAs of amylases with strong operational stability.
  • Higher affinity for substrate acquiring by NM-combi-CLEAs of amylases.
  • Higher thermostability representing by NM-combi-CLEAs of amylases.


Main Subjects

[1] Tomasik, P., Horton  D.(2012).Enzymatic Conversions of starch. Adv. Carbohydr. Chem. Biochem., 68,59-436.
[2] Romaškevič ,T., Budrienė ,S., Liubertienė, A., Gerasimčik ,I., Zubrienė ,A., Dienys, G.(2007).Synthesis of chitosan-graft-poly(ethylene glycol) methyl ether methacrylate copolymer and its application for immobilization of maltogenase. Chemija., 18 ,33–38.
[3] Bakker, M., van de Velde, F.,van Rantwijk ,F., Sheldon , R.A. (2000). Highly efficient  immobilization  of glycosylated enzymes into polyurethane foams.  Biotechnol. Bioeng.,70, 342–348.
[4] Straksys, A., Kochane,  T., Budriene,  S.(2016).Catalytic properties of maltogenic a-amylase from Bacillus stearothermophilus immobilized onto poly(urethane urea) microparticles. Food Chem., 294–299
[5] Demir,  A.,Topkaya,  R., Baykal, A. (2013).Green synthesis of superparamagnetic Fe3O4 nanoparticles with maltose: Its magnetic investigation. Polyhedron.,65,282-287.
[6] Saha,  B.C., Jordan,  D.B., Bothast,  R.J.(2009).Enzymes, Industrial (overview),3rd ed. Encyclopedia of Microbiology., 281-294.
[7] Talekar,  S., Pandharbale ,A., Ladole,  M., Nadar, S.H., Mulla, M., Japhalekar,  K., Pattankude, K., Arage,  D.( 2013). Carrier free co-immobilization of alpha amylase, glucoamylase and pullulanase as combined cross-linked enzyme aggregates (combi-CLEAs): a tri-enzyme biocatalyst with one pot starch hydrolytic activity. Bioresour. Technol.,147,269-275.
[8] Naadar, S.S., Rathod ,V.K.(2015).Magnetic macromolecular cross linked enzyme aggregates (CLEAs) of glucoamylase. Enzyme. Microb. Technol., 83, 78-87.
[9] Cao L., Langen L., Sheldon R.A.(2003). Immobilised enzymes: carrier-bound or carrier-free? Curr. Opin. Biotechnol.,14(4) ,387–394.
[10] Matijosˇyte, I., Arends, I.W.C.E., Vries,  S., Sheldon,  R.A.(2010). Preparation and use of cross-linked enzyme aggregates (CLEAs) of laccases. J. Mol. Catal. B: Enzym.,62,142–148.
[11] Talekar,  S., Ghodake , V., Ghotage,  T., Rathod,  P., Deshmukh,  P., Nadar,  S.H., Mulla,  M., Ladole,  M. (2012). Novel magnetic cross-linked enzyme aggregates (magnetic CLEAs) of alpha amylase. Bioresour. Technol., 123, 542–547.
[12] Torabizadeh, H., Mikani, M. (2018). Kinetic and thermodynamic features of nanomagnetic cross-linked enzyme aggregates of naringinase nanobiocatalyst in naringin hydrolysis, Int. J. Biol. Macromol., 119, 717–725.
 [13] Bhattacharya ,A. B., Pletschke, I. (2014) .Magnetic cross-linked enzyme aggregates (CLEAs): A novel concepttowards carrier free immobilization of lignocellulolytic enzymes. Enzyme. Microb. Technol., 61-62, 17–27
[14] Lu, A.H., Salabas ,E.L., Schüth, F.(2007). Magnetic nanoparticles: synthesis, protection, functionalization, and application. Angew. Chem. Int. Ed.,46 (8), 1222-44.
[15] Laurent ,S., Forge ,D., Port ,M., Roch, A., Robic, C., Elst, L.V.(2008). Magnetic iron oxide nanoparticles: synthesis, stabilization, vectorization, physicochemical characterizations, and biological applications. Chem. Rev., 108, 2064-2110.
[16] Cao, M., Wang ,J., Li ,Z., Ge ,W., Yue, T., Li ,R .( 2012). Food related applications of magnetic iron oxide nanoparticles: Enzyme immobilization, protein purification, and food analysis. Trends Food. Sci.Technol., 27, 47-56.
[17] Gupta ,A., Gupta, M. (2005). Synthesis and surface engineering of iron oxide nanoparticles for biomedical applications. Biomaterials., 26, 3995-4021.
[18] Schüth ,F.,Lu ,A.H.,Salbas, E.L. (2007). Magnetic Nanoparticles: Synthesis, Protection, Functionalization, and Application. Angew. Chem. Int. Ed., 46, 1222-1244.
[19] Reddy, L., Arias ,J., Nicolas, J., Couvreur ,P. (2012). Magnetic nanoparticles: Design and
Characterization, Toxicity and Biocompatibility. Pharmaceutical and Biomedical Applications . Chem. Rev., 112, 5818-5878.
[20] Berry ,C.C., Curtis ,A.S.G.(2003). Functionalisation of magnetic nanoparticles for applications in biomedicine. J. Phys. D: Appl. Phys., 36, 198-206.
[21] Cruz-Izquierdo,A., Pico´, E.A., Lo´pez, C., Serra, J.L., Liama,M. J. (2014). Magnetic Cross-Linked Enzyme Aggregates (mCLEAs) of Candida antarctica Lipase: An Efficient and Stable Biocatalyst for Biodiesel Synthesis. Biochem. Mol. Biol. Int., 436, 1145-1151.
[22] Liu ,W., Bai, S., Sun, Y.(2004). Preparation of nano-particles and its application in lipase  immobilization. J. Process .Eng., 4, 362-366.
[23] Khoshnevisan, K., Bordbar ,A.K., Zare, D., Davoodi ,D., Noruzi ,M., Barkhi, M.(2011). Immobilization of cellulase enzyme on superparamagnetic nanoparticles and determination of its activity and stability. Chem. Eng. J., 171, 669-673.
[24] Hsieh, H.C., Kuan ,I.C., Lee, S.L., Tien, G.Y., Wang, Y.J., Yu, C.Y.(2009). Stabilization of D-amino acid oxidase from Rhodosporidium toruloides by immobilization onto magnetic nanoparticles. Biotechnol. Lett., 31(4), 557-563.
[25] Namdeo, M., Bajpai ,S.K.(2009). Immobilization of a-amylase onto cellulose-coated magnetite (CCM) nanoparticles and preliminary starch degradation study. J. Mol. Catal. B: Enzym ., 59, 134-139.
[26] Torabizadeh ,H., Habibi-Rezaei, M., Safari, M., Moosavi-Movahedi ,A.A., Sharifizadeh ,A., Azizian, H., Amanlou ,M. (2011). Endo-inulinase stabilization by pyridoxal phosphate modification: A kinetics, thermodynamics, and simulation approach. Appl. Biochem. Biotechnol., 165, 1661-1673.
[27] Zia, M.,  Ali, A.,  Zafar, H., Haq, I.U.,  Phull ,A.R., Ali, J.S.,Hussain, A.( 2016).Synthesis, characterization, applications, and challenges of iron oxide nanoparticles. Nanotechnol. Sci. Appl., 9, 49-67.
[28] Couto, G., Klein. J., Schreiner .W., Mosca ,D., Oliveira ,A., Zarbin ,A. (2007). Nickel nanoparticles obtained by a modified polyol process: Synthesis, Characterization, and Magnetic Properties. J. Colloid Interface Sci., 311, 461-468.
[29] Bahmaie, M., Abbasi ,L., Faraji, M. (2013). Synthesis of magnetic nanoparticles (Fe3O4) and its application for extraction and preconcentration of drug sample from environmental samples. J. Semnan., 8, 29-37.
[30] Gao, Y., Kyratzis ,I. (2008). Covalent immobilization of proteins on carbon nanotubes using the cross-linker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide-A critical assessment. Bioconjugate Chem., 19, 1945-1950.
 [31] Nakamura, T., Ogata ,Y., Akichika ,S., Nakamura, A., Ohta ,K. (1995). Continuous production of fructose syrups from inulin by immobilized inulinase from Aspergillus niger mutant 817. J. Ferment. Bioeng., 80, 164-169.
[32] Missau, J., Scheid, A.J., Foletto ,E.L., Jahn ,S.L., Mazutti, M.A., Kuhn, R.C. (2014). Immobilization of commercial inulinase on alginate–chitosan beads. Enzyme Microb Technol., 2, 2-13.
[33] Torabizadeh, H., Tavakoli, M., Safari, M. (2014). Immobilization of thermostable Alpha-amylase from Bacillus licheniformis by cross-linked enzyme aggregates method using calcium and sodium ions as additives. J. Mol. Catal. B: Enzym., 108, 13-20.
[34] Bradford, M.M. (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.
[35] Viota, J.L., Arroyo, F.J., Delgado, A.V., Horno, J. (2010) Electrokinetic characterization of magnetite nanoparticles functionalized with amino acids. J. Colloid Interface Sci. 344, 144–149.
[36] Antal, I., Koneracka, M., Kubovcikova, M., Zavisova, V., Khmara, I., Lucanska, D., Jelenska, L., Vidlickova, I., Zatovicova, M., Pastorekova, S., Bugarova, N., Micusik, M.,
Omastova, M., Kopcansky, P. (2018). D,L-lysine functionalized Fe3O4 nanoparticles for detection of cancer cells. Colloids Surf. B: Biointerfaces. 163, 236–245.
[37] Vršanská, M., Vobˇerková, S., Jiménez Jiménez, A.M., Strmiska, V., Adam, V. (2017). Preparation and Optimisation of Cross-Linked Enzyme Aggregates Using Native Isolate White Rot Fungi Trametes versicolor and Fomes fomentarius for the Decolourisation of Synthetic Dyes. Int. J. Environ. Res. Public Health. 23, 1-15.
[38] Ribeiro, M.H.L., Rabaça, M. (2011). Cross-linked enzyme aggregates of naringinase: novel
biocatalysts for naringin hydrolysis. Enzyme Res., 2011, 1-8.
[39] Ramachandran, N., Hamborg, E.S., Versteeg, G.F. (2013). The effect of aqueous alcohols(methanol, t-butanol) and sulfolane on the dissociation constants and thermodynamic properties of alkanolamines. Fluid Phase Equilib., 360, 36–43.
[40] Sheldon, R.A., van Pelt, S. (2013). Enzyme immobilisation in biocatalysis: why, what and how. Chem. Soci. Rev., 42, 6223–6235.