Aspergillus oryzae S2 α- Amylase domain C involvement in activity and specificity: In vivo proteolysis, molecular and docking studies

Abstract

We previously reported that Aspergillus oryzae strain S2 had produced two α- Amylase isoforms named AmyA and AmyB. The apparent molecular masses revealed by SDS-PAGE were 50 and 42 kDa, respectively. Yet AmyB has a higher catalytic efficiency. Based on a monitoring study of the α- Amylase production in both the presence and absence of different protease inhibitors, a chymotrypsin proteolysis process was detected in vivo generating AmyB. A. oryzae S2 α- Amylase gene was amplified, cloned and sequenced. The sequence analysis revealed nine exons, eight introns and an encoding open reading frame of 1500 bp corresponding to AmyA isoform. The amino- Acid sequence analysis revealed aY371 potential chymotrypsin cleaving site, likely to be the AmyB C-Terminal end and two other potential sites at Y359, and F379. A zymogram with a high acrylamide concentration was used. It highlighted two other closed apparent molecular mass α- Amylases termed AmyB1 and AmyB2 reaching40 kDa and 43 kDa. These isoforms could be possibly generated fromY359, and,F379secondary cut, respectively. The molecular modeling study showed that AmyB preserved the (β/α)8 barrel domain and the domain B but lacked the C- Terminal domain C. The contact map analysis and the docking studies strongly suggested a higher activity and substrate binding affinity for AmyB than AmyA which was previously experimentally exhibited. This could be explained by the easy catalytic cleft accessibility.