Sahnoun Mouna, Jemli Sonia, Trabelsi Sahar, Ayadi Leila, Bejar Samir
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.
Preparatory Institute for Engineering Studies, Sfax (IPEIS), University of Sfax, MenzelChaker Road Km 0.5, P.O. Box 3018, Sfax, Tunisia.
PLoS One. 2016 Apr 21;11(4):e0153868. doi: 10.1371/journal.pone.0153868. eCollection 2016.
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.
我们之前报道过米曲霉S2菌株产生了两种α-淀粉酶同工型，分别命名为AmyA和AmyB。SDS-PAGE显示的表观分子量分别为50 kDa和42 kDa。然而，AmyB具有更高的催化效率。基于在存在和不存在不同蛋白酶抑制剂的情况下对α-淀粉酶产生的监测研究，在体内检测到了产生AmyB的胰凝乳蛋白酶蛋白水解过程。扩增、克隆并测序了米曲霉S2的α-淀粉酶基因。序列分析揭示了九个外显子、八个内含子以及一个对应于AmyA同工型的1500 bp编码开放阅读框。氨基酸序列分析揭示了一个Y371潜在胰凝乳蛋白酶切割位点，可能是AmyB的C末端，以及在Y359和F379处的另外两个潜在位点。使用了高丙烯酰胺浓度的酶谱。它突出显示了另外两种表观分子量相近的α-淀粉酶，分别称为AmyB1和AmyB2，分子量分别达到40 kDa和43 kDa。这些同工型可能分别由Y359和F379的二次切割产生。分子建模研究表明，AmyB保留了(β/α)8桶状结构域和结构域B，但缺少C末端结构域C。接触图分析和对接研究强烈表明，AmyB比AmyA具有更高的活性和底物结合亲和力，这与之前的实验结果一致。这可以通过催化裂隙易于接近来解释。
