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.

Functional Characterization of Recombinant Raw Starch Degrading α-Amylase from Roseateles terrae HL11 and Its Application on Cassava Pulp Saccharification

“…Regarding sequence similarity and phylogenetic distribution, interestingly, Gram-negative bacteria R. terrae HL11Amy, containing several typical animal motifs, is proposed as a new animal-like α-amylase with a starch-binding domain classified as CBM20, similar to Gram-positive bacteria T. fusca, Gram-negative bacteria Microbulbifer degradans and Actinobacteria Streptomyces limosus, probably resulting from repeated horizontal gene transfer from animals [40,41]. The alpha amylase C domain has been reported as a non-catalytic region important for the activity and starch-binding ability of several studied α-amylases [42,43]. Regarding sequence analysis, CBM20 contained two surface binding sites, including SBS1 and SBS2.…”

Apigenin isolated from A. americana encodes Human and Aspergillus oryzae S2 α-amylase inhibitions: credible approach for antifungal and antidiabetic therapies

“…The three-dimensional structure of Human pancreatic aamylase was imported from the Protein Data Bank (1HNY). The 3D structural model of the AmyA was generated using the SWISS-MODEL (http://www.expasy.org/ swissmod/) and the crystal structure of a-amylase from A. niger (PDB accession code 2GUY_A) as template which possesses 99% sequence identity with AmyA (Sahnoun et al 2016). Docking was performed with Autodock vina.…”

“…The three conserved domains of A. oryzae a-amylase were known as follow domain A (residues 1-122 and 181-383) domain B (residues 123-180) and domain C (residues 384-476). The catalytic A. oryzae catalytic triad consisted of Asp 206, Glu230, and Asp 297(Sahnoun et al 2016).…”

Bacillus velezensis Identification and Recombinant Expression, Purification, and Characterization of Its Alpha-Amylase

“…Alpha-amylase belongs to the glycoside hydrolase 13 (GH13) family, and most of them are Ca 2+ -dependent enzymes. The typical structure of α-amylase is composed of three domains: Domain A is in the N-terminal, composed of eight α-helices and eight β-strands linked by loops, forming an (α/β) 8 barrel; Domain B protrudes from the surface of the barrel and is located in the third α-helix and the third β-strand; Domain C is located at the C-terminal, composed of antiparallel β-strands [9,10].…”