Laboratory of Bioindustrial Genomics, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan.
Appl Microbiol Biotechnol. 2011 Dec;92(5):961-9. doi: 10.1007/s00253-011-3422-0. Epub 2011 Jun 18.
We observed that α-amylase (Taka-amylase A; TAA) activity in the culture broth disappeared in the later stage of submerged cultivation of Aspergillus oryzae. This disappearance was caused by adsorption of TAA onto the cell wall of A. oryzae and not due to protein degradation by extracellular proteolytic enzymes. To determine the cell wall component(s) that allows TAA adsorption efficiently, the cell wall was fractionated by stepwise alkali treatment and enzymatic digestion. Consequently, alkali-insoluble cell wall fractions exhibited high levels of TAA adsorption. In addition, this adsorption capacity was significantly enhanced by treatment of the alkali-insoluble fraction with β-glucanase, which resulted in the concomitant increase in the amount of chitin in the resulting fraction. In contrast, the adsorption capacity was diminished by treating the cell wall fraction with chitinase. These results suggest that the major component that allows TAA adsorption is chitin. However, both the mycelium and the cell wall demonstrated the inability to allow TAA adsorption in the early stage of cultivation, despite chitin content in the cell wall being identical in both early and late stages of cultivation. These results suggest the existence of unidentified factor(s) that could prevent the adsorption of TAA onto the cell wall. Such factor(s) is most likely removed or diminished from the cell wall following longer cultivation periods.
我们观察到，在米曲霉（Aspergillus oryzae）液体深层培养的后期，发酵液中的α-淀粉酶（Taka-amylase A；TAA）活性消失。这种消失是由于 TAA 被吸附到米曲霉细胞壁上，而不是由于细胞外蛋白酶对其进行的蛋白降解。为了确定允许 TAA 有效吸附的细胞壁成分，我们采用分步碱处理和酶消化的方法对细胞壁进行了分级。结果表明，碱不溶性细胞壁部分具有较高的 TAA 吸附能力。此外，用β-葡聚糖酶处理碱不溶性部分可显著提高这种吸附能力，同时导致该部分中几丁质的含量增加。相比之下，用几丁质酶处理细胞壁部分会降低其吸附能力。这些结果表明，允许 TAA 吸附的主要成分是几丁质。然而，在培养的早期阶段，菌丝体和细胞壁都不允许 TAA 吸附，尽管细胞壁中的几丁质含量在培养的早期和晚期阶段相同。这些结果表明，存在未知的因素可能会阻止 TAA 吸附到细胞壁上。这些因素很可能在较长的培养时间后从细胞壁中去除或减少。
