Electrospun Polyethersulfone Nanofibers: A Novel Matrix for Alpha-Amylase Immobilization

Document Type : Original Article


Department of Cell and Molecular Biology, Faculty of Biological Sciences, Kharazmi University, Tehran, Iran



Introduction: Amylases are used in various industries, mainly, starch processing that hydrolyze polysaccharides. Insoluble and solid supports are noteworthy in immobilization of enzymes for industry because of increasing enzyme stability. In this study, immobilization of alpha amylase in electrospun polyethersulfone (PES) nanofibers was studied.
Materials and Methods: Covalent immobilization of the enzyme was done through the carboxyl groups made by oxygen plasma treatment and 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) as a carboxyl group activator agent. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) and contact angle analysis proved enzyme immobilization. The optimum conditions determined and the catalytic parameters of immobilized enzyme were calculated.
Results: The results of this investigation showed that the optimum pH of immobilized enzyme was displaced toward acidic region by 1 unit. Comparison of the optimum temperature for immobilized and free amylase revealed 10°C increasing for the immobilized enzyme. Furthermore, the kinetic parameters, Vmax and Km for the immobilized enzyme were the same and higher than those of free ones, respectively. Storage stability of the immobilized amylase was obviously improved.
Conclusions: The results illustrated that nanofibrous supported alpha amylase is a new and suitable matrix for industrial applications.


  1. Singh S. A comparative study on immobilization of alpha amylase enzyme on different matricesIn. International Journal of Plant, Animal and Environmental Sciences. 2014;4(3):192-198.
  2. Singh P, Gupta P, Singh R, Sharma R. Activity and stability of immobilized alpha-amylase produced by Bacillus acidocaldarius. Int J Pharm Life Sci. 2012;3(12 Suppl):2247-2253.
  3. Raviyan P, Tang J, Rasco BA. Thermal stability of alpha-amylase from Aspergillus oryzae entrapped in polyacrylamide gel. J Agric Food Chem. 2003;51(18):5462-5466. doi:10.1021/jf020906j.
  4. Guisan JM. Immobilization of Enzymes and Cells. In: Brena BM, Batista-Viera F, eds. New York: Humana Press; 2006:15-30.
  5. Aksoy S, Tumturk H, Hasirci N. Stability of alpha-amylase immobilized on poly(methyl methacrylate-acrylic acid) microspheres. J Biotechnol. 1998;60(1-2):37-46.
  6. Bryjak J. Glucoamylase, α-amylase and β-amylase immobilisation on acrylic carriers. Biochem Eng J. 2003;16(3):347-355. doi:10.1016/S1369-703X(03)00114-1.
  7. Guiavarc’h Y, Van Loey A, Zuber F, Hendrickx M. Bacillus licheniformis α-amylase immobilized on glass beads and equilibrated at low moisture content: potentials as a Time– Temperature Integrator for sterilisation processes. Innov Food Sci Emerg Technol. 2004;5(3):317-325. doi:10.1016/j. ifset.2004.03.004.
  8. Kara A, Osman B, Yavuz H, Besirli N, Denizli A. Immobilization of α-amylase on Cu2+ chelated poly(ethylene glycol dimethacrylate-n-vinyl imidazole) matrix via adsorption. React Funct Polym. 2005;62(1):61-68. doi:10.1016/j.reactfunctpolym.2004.08.008.
  9. Konsoula Z, Liakopoulou-Kyriakides M. Starch hydrolysis by the action of an entrapped in alginate capsules α-amylase from Bacillus subtilis. Process Biochem. 2006;41(2):343-349. doi:10.1016/j. procbio.2005.01.028.
  10. Pandya PH, Jasra RV, Newalkar BL, Bhalt PN. Studies on the activity and stability of immobilized α-amylase in ordered mesoporous silicas. Microporous Mesoporous Mater. 2005;77(1):67-77. doi:10.1016/j.micromeso.2004.08.018.
  11. Mortazavi Milani Z, Jalal R, Goharshadi EK. Carbodiimide for Covalent α-Amylase Immobilization onto Magnetic Nanoparticles. Int J Nanosci. 2017;16(5-6):1750015. doi:10.1142/ s0219581x17500156.
  12. Ashly PC, Joseph MJ, Mohanan PV. Activity of diastase α-amylase immobilized on polyanilines (PANIs). Food Chem. 2011;127(4):1808-1813. doi:10.1016/j.foodchem.2011.02.068.
  13. Cordeiro AL, Lenk T, Werner C. Immobilization of Bacillus licheniformis alpha-amylase onto reactive polymer films. J Biotechnol. 2011;154(4):216-221. doi:10.1016/j. jbiotec.2011.04.008.
  14. Dey G, Nagpal V, Banerjee R. Immobilization of alpha-amylase from Bacillus circulans GRS 313 on coconut fiber. Appl Biochem Biotechnol. 2002;102-103(1-6):303-313.
  15. Kumari A, Kayastha AM. Immobilization of soybean (Glycine max) α-amylase onto Chitosan and Amberlite MB-150 beads: Optimization and characterization. J Mol Catal B Enzym. 2011;69(1-2):8-14. doi:10.1016/j.molcatb.2010.12.003.
  16. Namdeo M, Bajpai SK. Immobilization of α-amylase onto cellulose-coated magnetite (CCM) nanoparticles and preliminary starch degradation study. J Mol Catal B Enzym. 2009;59(1-3):134- 139. doi:10.1016/j.molcatb.2009.02.005.
  17. Soleimani M, Khani A, Najafzadeh K. α-Amylase immobilization on the silica nanoparticles for cleaning performance towards starch soils in laundry detergents. J Mol Catal B Enzym. 2012;74(1- 2):1-5. doi:10.1016/j.molcatb.2011.07.011.
  18. Datta S, Christena LR, Rajaram YR. Enzyme immobilization: an overview on techniques and support materials. 3 Biotech. 2013;3(1):1-9. doi:10.1007/s13205-012-0071-7.
  19. Basturk E, Demir S, Danıs O, Kahraman MV. Covalent immobilization of α-amylase onto thermally crosslinked electrospun PVA/PAA nanofibrous hybrid membranes. J Appl Polym Sci. 2013;127(1):349-355. doi:10.1002/app.37901.
  20. Oktay B, Demir S, Kayaman-Apohan N. Immobilization of alpha-amylase onto poly(glycidyl methacrylate) grafted electrospun fibers by ATRP. Mater Sci Eng C Mater Biol Appl. 2015;50:386- 393. doi:10.1016/j.msec.2015.02.033.
  21. Bernfeld P. Amylase α and β. In: Colowick SP, Kaplan NO, eds. Methods in Enzymology. New York: Academic Press Inc; 1955:149-158. Vol. 1. doi:10.1016/0076-6879(55)01021-5
  22. Miller GL. Use of dinitrosalicylic acid reagent for the determination of reducing sugar. Anal Chem. 1959;31(3):426-428. doi:10.1021/ ac60147a030.
  23. Dey TB, Kumar A, Banerjee R, Chandna P, Kuhad RC. Improvement of microbial α-amylase stability: Strategic approaches. Process Biochem. 2016;51(10):1380-1390. doi:10.1016/j. procbio.2016.06.021.
  24. Homaei A, Saberi D. Immobilization of α-amylase on gold nanorods: An ideal system for starch processing. Process Biochem. 2015;50(9):1394-1399. doi:10.1016/j.procbio.2015.06.002.
  25. Mohamad NR, Marzuki NH, Buang NA, Huyop F, Wahab RA. An overview of technologies for immobilization of enzymes and surface analysis techniques for immobilized enzymes. Biotechnol Biotechnol Equip. 2015;29(2):205-220. doi:10.1080/13102818.2 015.1008192.
  26. Tuzmen N, Kalburcu T, Denizli A. α-Amylase immobilization onto dye attached magnetic beads: Optimization and characterization. J Mol Catal B Enzym. 2012;78:16-23. doi:10.1016/j. molcatb.2012.01.017.
  27. Gupta MN, Kaloti M, Kapoor M, Solanki K. Nanomaterials as matrices for enzyme immobilization. Artif Cells Blood Substit Immobil Biotechnol. 2011;39(2):98-109. doi:10.3109/10731199 .2010.516259.
  28. Huang ZM, Zhang YZ, Kotaki M, Ramakrishna S. A review on polymer nanofibers by electrospinning and their applications in nanocomposites. Compos Sci Technol. 2003;63(15):2223-2253. doi:10.1016/S0266-3538(03)00178-7.
  29. Alenazi NA, Hussein MA, Alamry KA, Asiri AM. Modified polyether-sulfone membrane: a mini review. Des Monomers Polym. 2017;20(1):532-546. doi:10.1080/15685551.2017.1398 208.
  30. Li Y, Quan J, Branford-White C, Williams GR, Wu JX, Zhu LM. Electrospun polyacrylonitrile-glycopolymer nanofibrous membranes for enzyme immobilization. J Mol Catal B Enzym. 2012;76:15-22. doi:10.1016/j.molcatb.2011.12.003.
  31. Kazenwadel F, Wagner H, Rapp BE, Franzreb M. Optimization of enzyme immobilization on magnetic microparticles using 1-ethyl- 3-(3-dimethylaminopropyl)carbodiimide (EDC) as a crosslinking agent. Anal Methods. 2015;7(24):10291-10298. doi:10.1039/ C5AY02670A.
  32. Ge L, Zhao YS, Mo T, Li JR, Li P. Immobilization of glucose oxidase in electrospun nanofibrous membranes for food preservation. Food Control. 2012;26(1):188-193. doi:10.1016/j. foodcont.2012.01.022.
  33. Ghosh S, Chaganti SR, Prakasham RS. Polyaniline nanofiber as a novel immobilization matrix for the anti-leukemia enzyme l-asparaginase. J Mol Catal B Enzym. 2012;74(1-2):132-137. doi:10.1016/j.molcatb.2011.09.009
  34. Kahraman MV, Bayramoglu G, Kayaman-Apohan N, Gungor A. α-Amylase immobilization on functionalized glass beads by covalent attachment. Food Chem. 2007;104(4):1385-1392. doi:10.1016/j.foodchem.2007.01.054
  35. Dwevedi A. Enzyme Immobilization: Advances in Industry, Agriculture, Medicine, and the Environment. Switzerland: Springer; 2016.
  36. Bisswanger H. Enzyme assays. Perspect Sci. 2014;1(1-6):41-55. doi:10.1016/j.pisc.2014.02.005.
  37. Bayramoglu Z, Akbulut U, Sungur S. Immobilization of alpha-amylase into photographic gelatin by chemical cross-linking. Biomaterials. 1992;13(10):704-708.
  38. Arica MY, Hasirci V, Alaeddinoglu NG. Covalent immobilization of alpha-amylase onto pHEMA microspheres: preparation and application to fixed bed reactor. Biomaterials. 1995;16(10):761- 768.
  39. Wang ZG, Ke BB, Xu ZK. Covalent immobilization of redox enzyme on electrospun nonwoven poly(acrylonitrile-co-acrylic acid) nanofiber mesh filled with carbon nanotubes: a comprehensive study. Biotechnol Bioeng. 2007;97(4):708-720. doi:10.1002/bit.21280.
  40. Lee PM, Lee KH, Siaw YS. Covalent immobilization of aminoacylase to alginate for L-phenylalanine production. J Chem Technol Biotechnol. 1993;58(1):65-70. doi:10.1002/jctb.280580109.
  41. Demircioglu H, Beyenal H, Tanyolac A, Hasirci N. Immobilization of urease and estimation of effective diffusion coefficients of urea in HEMA and VP copolymer matrices. Polym Int. 1994;35(4):321- 327. doi:10.1002/pi.1994.210350404.
  42. El-Aassar MR, Al-Deyab SS, Kenawy ER. Covalent immobilization of β-galactosidase onto electrospun nanofibers of poly (AN-co- MMA) copolymer. J Appl Polym Sci. 2013;127(3):1873-1884. doi:10.1002/app.37922.
  43. El-Aassar MR. Functionalized electrospun nanofibers from poly (AN-co-MMA) for enzyme immobilization. J Mol Catal B Enzym. 2013;85-86:140-148. doi:10.1016/j.molcatb.2012.09.002.
  44. Singh K, Srivastava G, Talat M, Srivastava ON, Kayastha AM. α-Amylase immobilization onto functionalized graphene nanosheets as scaffolds: Its characterization, kinetics and potential applications in starch based industries. Biochem Biophys Rep. 2015;3:18-25. doi:10.1016/j.bbrep.2015.07.002.