In this study, we report the identification and characterization of an acetyl xylan esterase, designated as AoAXEC, which was previously annotated as a hypothetical protein encoded by AO090023000158 in the Aspergillus oryzae genomic database. Based on its amino acid sequence, a low sequence identity to known acetyl xylan esterases was observed in the sequence of characterized acetyl xylan esterase. The gene fused with α-factor signal sequence of Saccharomyces cerevisiae instead of the native signal sequence was cloned into a vector, pPICZαC, and expressed successfully in Pichia pastoris as an active extracellular protein. The purified recombinant protein had pH and temperature optima of 7.0 and 50 °C, respectively, and was stable up to 50 °C. The optimal substrate for hydrolysis by the purified recombinant AoAXEC, among a panel of α-naphthyl esters (C2–C16), was α-naphthyl propionate (C3), with an activity of 0.35 ± 0.006 units/mg protein. No significant difference of the K_m value was observed between C3 (2.3 ± 0.7 mM) and C2 (1.9 ± 0.4 mM). In contrast, k_cat value for C3 (18 ± 3.9 s⁻¹) was higher compared to C2 (4.5 ± 0.7 s⁻¹). The purified recombinant enzyme displayed a low activity toward acyl chain substrates containing eight or more carbon atoms. Recombinant AoAXEC catalyzed the release of acetic acid from wheat arabinoxylan. However, no activity was detected on methyl esters of ferulic, p-coumaric, caffeic, or sinapic acids. Additionally, the liberation of phenolic acids, such as ferulic acid, from wheat arabinoxylan was not exhibited by the recombinant protein.

在这项研究中，我们报告了一种乙酰木聚糖酯酶的鉴定和表征，称为Ao AXEC，它以前在米曲霉基因组数据库中被注释为由 AO090023000158 编码的假设蛋白质。基于其氨基酸序列，在表征的乙酰木聚糖酯酶的序列中观察到与已知乙酰木聚糖酯酶的低序列同一性。将与酿酒酵母α因子信号序列而非天然信号序列融合的基因克隆到载体pPICZαC中，并在毕赤酵母中成功表达作为一种活性细胞外蛋白。纯化的重组蛋白的最适 pH 值和温度分别为 7.0 和 50 °C，并在 50 °C 下保持稳定。在一组α-萘酯（C2-C16）中，纯化的重组Ao AXEC水解的最佳底物是丙酸α-萘酯（C3），其活性为0.35 ± 0.006 单位/mg 蛋白质。在 C3 (2.3 ± 0.7 mM) 和 C2 (1.9 ± 0.4 mM) 之间没有观察到K _m值的显着差异。相比之下，C3 (18 ± 3.9 s ^-1 ) 的k _cat值高于 C2 (4.5 ± 0.7 s ^-1 )。纯化的重组酶对含有八个或更多碳原子的酰基链底物显示出低活性。重组Ao AXEC 催化从小麦阿拉伯木聚糖中释放乙酸。然而，对阿魏酸、对香豆酸、咖啡酸或芥子酸的甲酯没有检测到活性。此外，重组蛋白没有表现出从小麦阿拉伯木聚糖中释放酚酸，例如阿魏酸。