Ruminiclostridium josui Fae1A is a modular enzyme consisting of an N-terminal signal peptide, family-1 carbohydrate esterase module (CE1), family-6 carbohydrate-binding module (CBM6), and dockerin module in that order. Recombinant CE1 and CBM6 polypeptides were collectively and separately produced as RjFae1A, RjCE1, and RjCBM6. RjFae1A showed higher feruloyl esterase activity than RjCE1 towards insoluble wheat arabinoxylan, but the latter was more active towards small synthetic substrates than the former. This suggests that CBM6 in RjFae1A plays an important role in releasing ferulic acid from the native substrate. RjCBM6 showed a higher affinity for soluble wheat arabinoxylan than for rye arabinoxylan and beechwood xylan in native affinity polyacrylamide gel electrophoresis. Isothermal titration calorimetry analysis demonstrated that RjCBM6 recognized a xylopyranosyl residue at the nonreducing ends of xylooligosaccharides. Moreover, it showed exceptional affinity for 23-α-l-arabinofuranosyl-xylotriose (A2XX) among the tested branched arabinoxylooligosaccharides. Fluorometric titration analysis demonstrated that xylobiose and A2XX competitively bound to RjCBM6, and both bound to the same site in RjCBM6. RjCBM6's preference for the xylopyranosyl residue at the nonreducing end of xylan chains explains why the positive effect of CBM6 on RjFae1A activity was observed only during short incubation but not after extended incubation.

中文翻译：

---

模块化阿魏酸酯酶中家族 6 碳水化合物结合模块用于从不溶性小麦阿拉伯木聚糖中去除阿魏酸的意义

Ruminiclostridium josui Fae1A 是一种模块化酶，由 N 端信号肽、家族 1 碳水化合物酯酶模块 (CE1)、家族 6 碳水化合物结合模块 (CBM6) 和 dockerin 模块组成。重组 CE1 和 CBM6 多肽作为 RjFae1A、RjCE1 和 RjCBM6 共同和单独产生。RjFae1A 对不溶性小麦阿拉伯木聚糖显示出比 RjCE1 更高的阿魏酸酯酶活性，但后者对小合成底物的活性高于前者。这表明 RjFae1A 中的 CBM6 在从天然底物释放阿魏酸中起重要作用。在天然亲和聚丙烯酰胺凝胶电泳中，RjCBM6 对可溶性小麦阿拉伯木聚糖的亲和力高于黑麦阿拉伯木聚糖和山毛榉木聚糖。等温滴定量热分析表明，RjCBM6 在低聚木糖的非还原末端识别了吡喃木糖残基。此外，它在测试的支链阿拉伯木寡糖中显示出对 23-α-l-阿拉伯呋喃糖基木三糖 (A2XX) 的特殊亲和力。荧光滴定分析表明，木二糖和 A2XX 与 RjCBM6 竞争结合，并且都与 RjCBM6 中的同一位点结合。RjCBM6 对木聚糖链非还原末端的吡喃木糖残基的偏爱解释了为什么 CBM6 对 RjFae1A 活性的积极影响仅在短时间孵育期间观察到，而在延长孵育时间后观察不到。在测试的支链阿拉伯木寡糖中，它对 23-α-l-阿拉伯呋喃糖基木三糖 (A2XX) 显示出非凡的亲和力。荧光滴定分析表明，木二糖和 A2XX 与 RjCBM6 竞争结合，并且都与 RjCBM6 中的同一位点结合。RjCBM6 对木聚糖链非还原末端的吡喃木糖残基的偏爱解释了为什么 CBM6 对 RjFae1A 活性的积极影响仅在短时间孵育期间观察到，而在延长孵育时间后观察不到。在测试的支链阿拉伯木寡糖中，它对 23-α-l-阿拉伯呋喃糖基木三糖 (A2XX) 显示出非凡的亲和力。荧光滴定分析表明，木二糖和 A2XX 与 RjCBM6 竞争结合，并且都与 RjCBM6 中的同一位点结合。RjCBM6 对木聚糖链非还原末端的吡喃木糖残基的偏爱解释了为什么 CBM6 对 RjFae1A 活性的积极影响仅在短时间孵育期间观察到，而在延长孵育时间后观察不到。