Sahnoun Mouna, Kriaa Mouna, Elgharbi Fatma, Ayadi Dorra-Zouari, Bejar Samir, Kammoun Radhouane
Laboratory of Microorganisms and Biomolecules (LMB), Centre of Biotechnology of Sfax (CBS), University of Sfax, Road of Sidi Mansour Km 6, PO Box 1177, Sfax 3018, Tunisia.
Laboratory of Microorganisms and Biomolecules (LMB), Centre of Biotechnology of Sfax (CBS), University of Sfax, Road of Sidi Mansour Km 6, PO Box 1177, Sfax 3018, Tunisia.
Int J Biol Macromol. 2015 Apr;75:73-80. doi: 10.1016/j.ijbiomac.2015.01.026. Epub 2015 Jan 21.
Aspergillus oryzae S2 was assayed for alpha-amylase production under solid state fermentation (SSF). In addition to AmyA and AmyB already produced in monitored submerged culture, the strain was noted to produce new AmyB oligomeric forms, in particular a dominant tetrameric form named AmyC. The latter was purified to homogeneity through fractional acetone precipitation and size exclusion chromatography. SDS-PAGE and native PAGE analyses revealed that, purified AmyC was an approximately 172 kDa tetramer of four 42 kDa subunits. AmyC was also noted to display the same NH2-terminal amino acid sequence residues and approximately the same physico-chemical properties of AmyA and AmyB, to exhibit maximum activity at pH 5.6 and 60 °C, and to produce maltose and maltotriose as major starch hydrolysis end-products. Soyabean meal was the best substitute to yeast extract compared to fish powder waste and wheat gluten waste. AmyC production was optimized under SSF using statistical design methodology. Moisture content of 76.25%, C/N substrate ratio of 0.62, and inoculum size of 10(6.87) spores allowed maximum activity of 22118.34 U/g of dried substrate, which was 33 times higher than the one obtained before the application of the central composite design (CCD).
对米曲霉S2进行固态发酵（SSF）产α-淀粉酶的测定。除了在监测的液体培养中已产生的AmyA和AmyB外，该菌株还产生了新的AmyB寡聚体形式，特别是一种主要的四聚体形式，命名为AmyC。通过分级丙酮沉淀和尺寸排阻色谱法将后者纯化至同质。SDS-PAGE和非变性PAGE分析表明，纯化后的AmyC是由四个42 kDa亚基组成的约172 kDa的四聚体。还发现AmyC与AmyA和AmyB具有相同的NH2末端氨基酸序列残基以及大致相同的物理化学性质，在pH 5.6和60°C时表现出最大活性，并产生麦芽糖和麦芽三糖作为淀粉水解的主要终产物。与鱼粉废料和小麦面筋废料相比，豆粕是酵母提取物的最佳替代品。使用统计设计方法在固态发酵条件下对AmyC的生产进行了优化。水分含量76.25%、C/N底物比0.62和接种量10(6.87)个孢子时，每克干底物的最大活性为22118.34 U，这比应用中心复合设计（CCD）之前获得的活性高33倍。
