A three catalytic domain multi-enzyme; a CE1 ferulic acid esterase, a GH62 α-l-arabinofuranosidase and a GH10 β-d-1,4-xylanase was identified in a metagenome obtained from wastewater treatment sludge. The capability of the CE1-GH62-GH10 multi-enzyme to degrade arabinoxylan was investigated to examine the hypothesis that CE1-GH62-GH10 would degrade arabinoxylan more efficiently than the corresponding equimolar mix of the individual enzymes. CE1-GH62-GH10 efficiently catalyzed the production of xylopyranose, xylobiose, xylotriose, arabinofuranose and ferulic acid (FA) when incubated with insoluble wheat arabinoxylan (WAX-I) (kcat = 20.8 ± 2.6 s-1). Surprisingly, in an equimolar mix of the individual enzymes a similar kcat towards WAX-I was observed (kcat = 17.3 ± 3.8 s-1). Similarly, when assayed on complex plant biomass the activity was comparable between CE1-GH62-GH10 and an equimolar mix of the individual enzymes. This suggests that from a hydrolytic point of view a CE1-GH62-GH10 multi-enzyme is not an advantage. Determination of the melting temperatures for CE1-GH62-GH10 (71.0 ± 0.05 °C) and CE1 (69.9 ± 0.02), GH62 (65.7 ± 0.06) and GH10 (71 ± 0.05 °C) indicates that CE1 and GH62 are less stable as single domain enzymes. This conclusion was corroborated by the findings that CE1 lost ˜50% activity within 2 h, while GH62 retained ˜50% activity after 24 h, whereas CE1-GH62-GH10 and GH10 retained ˜50% activity for 72 h. GH62-GH10, when appended to each other, displayed a higher specificity constant (kcat/Km = 0.3 s-1 mg-1 ml) than the individual GH10 (kcat/Km = 0.12 s-1 ± 0.02 mg-1 ml) indicating a synergistic action between the two. Surprisingly, CE1-GH62, displayed a 2-fold lower kcat towards WAX-I than GH62, which might be due to the presence of a putative carbohydrate binding module appended to CE1 at the N-terminal. Both CE1 and CE1-GH62 released insignificant amounts of FA from WAX-I, but FA was released from WAX-I when both CE1 and GH10 were present, which might be due to GH10 releasing soluble oligosaccharides that CE1 can utilize as substrate. CE1 also displayed activity towards solubilized 5-O-trans-feruloyl-α-l-Araf (kcat = 36.35 s-1). This suggests that CE1 preferably acts on soluble oligosaccharides.

中文翻译：

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针对阿魏酰化阿拉伯木聚糖降解的新型木聚糖分解三域酶

一种三催化域多酶；在从废水处理污泥中获得的宏基因组中鉴定了 CE1 阿魏酸酯酶、GH62 α-l-阿拉伯呋喃糖苷酶和 GH10 β-d-1,4-木聚糖酶。研究了 CE1-GH62-GH10 多酶降解阿拉伯木聚糖的能力，以检验 CE1-GH62-GH10 比相应的单个酶的等摩尔混合物更有效地降解阿拉伯木聚糖的假设。CE1-GH62-GH10 与不溶性小麦阿拉伯木聚糖 (WAX-I) (kcat = 20.8 ± 2.6 s-1) 一起孵育时，可有效催化吡喃木糖、木二糖、木三糖、阿拉伯呋喃糖和阿魏酸 (FA) 的产生。令人惊讶的是，在单个酶的等摩尔混合物中，观察到与 WAX-I 相似的 kcat (kcat = 17.3 ± 3.8 s-1)。相似地，当对复杂的植物生物量进行分析时，CE1-GH62-GH10 和单个酶的等摩尔混合物之间的活性相当。这表明从水解的角度来看，CE1-GH62-GH10 多酶不是优势。确定 CE1-GH62-GH10 (71.0 ± 0.05 °C) 和 CE1 (69.9 ± 0.02)、GH62 (65.7 ± 0.06) 和 GH10 (71 ± 0.05 °C) 的熔化温度表明 CE1 和 GH62 的稳定性较差单域酶。CE1 在 2 小时内失去了约 50% 的活性，而 GH62 在 24 小时后保留了约 50% 的活性，而 CE1-GH62-GH10 和 GH10 在 72 小时内保留了约 50% 的活性，这一发现证实了这一结论。GH62-GH10 相互附加时，显示出比单独的 GH10（kcat/Km = 0.12 s-1 ± 0）更高的特异性常数（kcat/Km = 0.3 s-1 mg-1 ml）。02 mg-1 ml) 表明两者之间的协同作用。令人惊讶的是，CE1-GH62 对 WAX-1 的 kcat 比 GH62 低 2 倍，这可能是由于在 N 端附加到 CE1 的推定碳水化合物结合模块的存在。CE1 和 CE1-GH62 都从 WAX-I 中释放出微量的 FA，但是当 CE1 和 GH10 都存在时，FA 从 WAX-I 中释放出来，这可能是由于 GH10 释放了 CE1 可以用作底物的可溶性寡糖。CE1 还显示出对溶解的 5-O-反式阿魏酰-α-l-Araf (kcat = 36.35 s-1) 的活性。这表明 CE1 优先作用于可溶性寡糖。CE1 和 CE1-GH62 都从 WAX-I 中释放出微量的 FA，但是当 CE1 和 GH10 都存在时，FA 从 WAX-I 中释放出来，这可能是由于 GH10 释放了 CE1 可以用作底物的可溶性寡糖。CE1 还显示出对溶解的 5-O-反式阿魏酰-α-l-Araf (kcat = 36.35 s-1) 的活性。这表明 CE1 优先作用于可溶性寡糖。CE1 和 CE1-GH62 都从 WAX-I 中释放出微量的 FA，但是当 CE1 和 GH10 都存在时，FA 从 WAX-I 中释放出来，这可能是由于 GH10 释放了 CE1 可以用作底物的可溶性寡糖。CE1 还显示出对溶解的 5-O-反式阿魏酰-α-l-Araf (kcat = 36.35 s-1) 的活性。这表明 CE1 优先作用于可溶性寡糖。