Immobilization of fenugreek β-amylase onto functionalized graphene quantum dots (GQDs) using Box-Behnken design: Its biochemical, thermodynamic and kinetic studies

Int J Biol Macromol. 2020 Feb 1:144:170-182. doi: 10.1016/j.ijbiomac.2019.12.033. Epub 2019 Dec 13.

Abstract

β-Amylase was immobilized onto GQDs using 3-aminopropyltriethoxysilane and glutaraldehyde. Optimization was carried out by Box-Behnken design and binding was confirmed by SEM, AFM, FTIR and fluorescence microscopy. Predicted optimum immobilization efficiency (88.64%) was very close to actual (87.98%), which confirmed the success of the immobilization process. The immobilized enzyme showed maximum activity at pH 5.0 and 57 °C, whereas Km and Vmax were found to be 6.40 mg/mL and 714.28 μmol/min/mg, respectively. The enzyme retained 75% activity after 12 uses at 30 °C. Increased values of ΔG° ΔH°, half-life and activation energy of the enzyme inactivation (ΔEd) revealed that thermo-stability increases after immobilization and the process followed first-order kinetics (r2 > 0.96). The activation energy of catalysis (ΔEa) and ΔEd for immobilized enzyme were 22.58 and 158.99 ± 1.10 kJ/mol, respectively which revealed that denaturation of the enzyme requires a higher amount of energy rather than catalysis. Thermodynamic and fluorescence spectroscopic studies revealed that the process is non-spontaneous (ΔG > 0) and endothermic (ΔH > 0) and occurred through protein unfolding rather than aggregation (ΔS > 0). Thus increase in thermo-stability of immobilized fenugreek β-amylase and non-toxic nature of GQDs could be exploited for maltose production in beverage, food and pharmaceutical industries.

Keywords: Gibbs free energy; Immobilization; β-Amylase.

MeSH terms

  • Enzyme Stability
  • Enzymes, Immobilized / metabolism*
  • Germination
  • Graphite / chemistry*
  • Hydrogen-Ion Concentration
  • Kinetics
  • Quantum Dots / chemistry*
  • Quantum Dots / ultrastructure
  • Solubility
  • Spectroscopy, Fourier Transform Infrared
  • Thermodynamics
  • Trigonella / enzymology*
  • beta-Amylase / metabolism*

Substances

  • Enzymes, Immobilized
  • Graphite
  • beta-Amylase