Aspergillus Oryzae S2 α-Amylase Domain C Involvement in Activity and Specificity: In Vivo Proteolysis, Molecular and Docking Studies.
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.
Laboratory of Microbial Biotechnology and Engineering Enzymes (LMBEE), Centre of Biotechnology of Sfax (CBS), University of Sfax, Sidi Mansour Road Km 6, P.O. Box 1177, Sfax, 3018, Tunisia.
Mouna
M
Sahnoun
Laboratory of Microbial Biotechnology and Engineering Enzymes (LMBEE), Centre of Biotechnology of Sfax (CBS), University of Sfax, Sidi Mansour Road Km 6, P.O. Box 1177, Sfax, 3018, Tunisia.
Sonia
S
Jemli
Laboratory of Microbial Biotechnology and Engineering Enzymes (LMBEE), Centre of Biotechnology of Sfax (CBS), University of Sfax, Sidi Mansour Road Km 6, P.O. Box 1177, Sfax, 3018, Tunisia.
Sahar
S
Trabelsi
Preparatory Institute for Engineering Studies, Sfax (IPEIS), University of Sfax, MenzelChaker Road Km 0.5, P.O. Box 3018, Sfax, Tunisia.
Leila
L
Ayadi
Laboratory of Microbial Biotechnology and Engineering Enzymes (LMBEE), Centre of Biotechnology of Sfax (CBS), University of Sfax, Sidi Mansour Road Km 6, P.O. Box 1177, Sfax, 3018, Tunisia.
Samir
S
Bejar
D000595:Amino Acid Sequence; D001236:Aspergillus oryzae / Q000201:enzymology*; D062105:Molecular Docking Simulation; D059748:Proteolysis; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D000516:alpha-Amylases / Q000737:chemistry / Q000378:metabolism*
D000516:alpha-Amylases