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

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 (β3-β4) and (β7-β8) 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.