RESEARCH ARTICLE Aspergillus Oryzae S2 α-Amylase Domain C Involvement in Activity and Specificity: In Vivo Proteolysis, Molecular and Docking Studies Mouna Sahnoun 1 , Sonia Jemli 1 , Sahar Trabelsi 1 , Leila Ayadi 2 , Samir Bejar 1 * 1 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, 2 Preparatory Institute for Engineering Studies, Sfax (IPEIS), University of Sfax, MenzelChaker Road Km 0.5, P.O. Box 3018, Sfax, Tunisia * samir.bejar@cbs.rnrt.tn Abstract We previously reported that Aspergillus oryzae strain S2 had produced two α-amylase iso- forms 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 AmyB 1 and AmyB 2 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 experimen- tally exhibited. This could be explained by the easy catalytic cleft accessibility. Introduction Amylases cover about 25 to33% of the world enzyme market [1]. They are used in several industries mainly in the hydrolysis of starch to generate glucose, maltose, a mixture of malto- oligosaccharides and α-limit dextrin-containing α-(1–6) bonds [2]. Those products are highly important in a wide range of nutritional, cosmetic and pharmaceutical applications [3–5].α- amylases were classified into α-1, 4-glucan-4-glucanohydrolase, EC 3.2.1.1 [6] according to PLOS ONE | DOI:10.1371/journal.pone.0153868 April 21, 2016 1 / 15 a11111 OPEN ACCESS Citation: Sahnoun M, Jemli S, Trabelsi S, Ayadi L, Bejar S (2016) Aspergillus Oryzae S2 α-Amylase Domain C Involvement in Activity and Specificity: In Vivo Proteolysis, Molecular and Docking Studies. PLoS ONE 11(4): e0153868. doi:10.1371/journal. pone.0153868 Editor: Luis Menéndez-Arias, Centro de Biología Molecular Severo Ochoa (CSIC-UAM), SPAIN Received: November 13, 2015 Accepted: April 5, 2016 Published: April 21, 2016 Copyright: © 2016 Sahnoun et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by a grant from the Tunisian Ministry of Higher Education and Scientific Research contract program CBSLMBEE/ code: LR15CBS06_2015-2018. There was no additional external funding received for this study. Competing Interests: The authors have declared that no competing interests exist.