Modifing Aspergillus Oryzae S2 amylase substrate specificity and thermostability through its tetramerisation using biochemical and in silico studies and stabilization

Int J Biol Macromol. 2018 Oct 1;117:483-492. doi: 10.1016/j.ijbiomac.2018.05.136. Epub 2018 May 23.


We previously reported that Aspergillus oryzae S2 had produced an amylase called AmyC formed by a tetramer of AmyB subunits under solid state fermentation. In this work, we demonstrated that the half-life time of AmyC at 75 °C and 80 °C were remarkably enhanced to reach 53 min and 41 min compared to 6 min and 4 min for AmyB. The Km values of AmyC for maltoheptaose, maltopentaose, and maltotetraose were 2-fold lower than AmyB. AmyC showed a 6.5 fold higher exo-type activity and hydrolyzed the short oligosaccharides more efficiently than AmyB. The AmyC-3D model was generated and showed that a region named T1 was involved in the oligomerization process. The subunits and the RING network interactions insight suggested that AmyC sub-units were bounded by 20 hydrogen bonds, 4 electrostatic interactions, 16 nodes and 836 edges leading to a higher thermal stability. The disordered (β34) and (β78) loops contained in the AmyC active cleft were presumed to be the recognition sites of the non-reducing end substrate. The docking studies strongly suggested that AmyC easily accommodated the short substrates as it was exhibited in vitro and seemed to look like maltogenic amylases. The Box-Behnken Response Surface Methodology was applied for Amy C immobilization for efficient use. An optimum condition of an aluminum oxide content of 0.25 g, a carrageenan content of 0.1 g, and a glutaraldehyde content of 0.5%/g of carrier resulted in 76.2% of covalent immobilization yield. The immobilized AmyC kept its total activity for three cycles, shifted the optimum temperature from 60 °C to 65 °C, and had two-fold half-life at 85 °C compared to the free enzyme.

Keywords: 3D-model; AmyB; AmyC; Maltogenic amylase; Stabilization; Substrate selectivity; Tetrameric form; Thermostability.

MeSH terms

  • Amylases / metabolism*
  • Aspergillus oryzae / enzymology*
  • Binding Sites
  • Computer Simulation*
  • Enzyme Stability
  • Hydrolysis
  • Kinetics
  • Models, Molecular
  • Protein Multimerization*
  • Protein Subunits / metabolism
  • Spectroscopy, Fourier Transform Infrared
  • Static Electricity
  • Substrate Specificity
  • Temperature*


  • Protein Subunits
  • Amylases