Abstract

Arabinofuranose substitutions on xylan are known to interfere with enzymatic hydrolysis of this primary hemicellulose. In this work, two novel α-l-arabinofuranosidases (ABFs), TtABF51A from Thielavia terrestris and EpABF62C from Eupenicillium parvum, were characterized and functionally analyzed. From sequences analyses, TtABF51A and EpABF62C belong to glycoside hydrolase (GH) families 51 and 62, respectively. Recombinant TtABF51A showed high activity on 4-nitrophenyl-α-l-arabinofuranoside (83.39 U/mg), low-viscosity wheat arabinoxylan (WAX, 39.66 U/mg), high-viscosity rye arabinoxylan (RAX, 32.24 U/mg), and sugarbeet arabinan (25.69 U/mg), while EpABF62C preferred to degrade arabinoxylan. For EpABF62C, the rate of hydrolysis of RAX (94.10 U/mg) was 2.1 times that of WAX (45.46 U/mg). The optimal pH and reaction temperature for the two enzymes was between 4.0 and 4.5 and 65 °C, respectively. Calcium played an important role in the thermal stability of EpABF62C. TtABF51A and EpABF62C showed the highest thermal stabilities at pH 4.5 or 5.0, respectively. At their optimal pHs, TtABF51A and EpABF62C retained greater than 80% of their initial activities after incubation at 55 °C for 96 h or 144 h, respectively. ¹H NMR analysis indicated that the two enzymes selectively removed arabinose linked to C-3 of mono-substituted xylose residues in WAX. Compared with the singular application of the GH10 xylanase EpXYN1 from E. parvum, co-digestions of WAX including TtABF51A and/or EpABF62C released 2.49, 3.38, and 4.81 times xylose or 3.38, 1.65, and 2.57 times of xylobiose, respectively. Meanwhile, the amount of arabinose released from WAX by TtABF51A with EpXYN1 was 2.11 times the amount with TtABF51A alone.

Key points

• Two novel α- l -arabinofuranosidases (ABFs) displayed high thermal stability.

• The thermal stability of GH62 family EpABF62C was dependent on calcium.

• Buffer pH affects the thermal stability of the two ABFs.

• Both ABFs enhance the hydrolysis of WAX by a GH10 xylanase.

中文翻译：

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两种新的耐热性α-L-阿拉伯呋喃糖苷酶属于地衣藻（Thielavia terrestris）的糖苷水解酶家族51和小叶杜仲（Eupenicillium parvum）的家族62。

摘要

已知木聚糖上的阿拉伯呋喃糖取代会干扰该初级半纤维素的酶促水解。在这项工作中，对两种新颖的α- 1-阿拉伯呋喃糖苷酶（ABFs），来自地衣藻（Thielavia terrestris）的TtABF51A和来自小叶杜仲（Eupenicillium parvum）的EpABF62C进行了表征和功能分析。从序列分析中，TtABF51A和EpABF62C分别属于糖苷水解酶（GH）家族51和62。重组TtABF51A对4-硝基苯基-α- 1具有高活性-阿拉伯呋喃糖苷（83.39 U / mg），低粘度小麦阿拉伯木聚糖（WAX，39.66 U / mg），高粘度黑麦阿拉伯木聚糖（RAX，32.24 U / mg）和甜菜阿拉伯聚糖（25.69 U / mg），而EpABF62C优选降解阿拉伯木聚糖。对于EpABF62C，RAX（94.10 U / mg）的水解速率是WAX（45.46 U / mg）的2.1倍。两种酶的最佳pH和反应温度分别在4.0至4.5和65°C之间。钙在EpABF62C的热稳定性中起重要作用。TtABF51A和EpABF62C分别在pH 4.5或5.0时显示出最高的热稳定性。在其最佳pH值下，TtABF51A和EpABF62C在55°C孵育96 h或144 h后，分别保留了其初始活性的80％以上。^1个1 H NMR分析表明，这两种酶选择性地除去了WAX中与单取代木糖残基的C-3连接的阿拉伯糖。与GH10的单数应用木聚糖酶相比，从EpXYN1 E.孢子虫分别WAX的共消化包括TtABF51A和/或释放EpABF62C 2.49，3.38，和木二糖的4.81倍木糖或3.38，1.65，和2.57倍。同时，TpABF51A与EpXYN1从WAX释放的阿拉伯糖量是单独TtABF51A的2.11倍。

关键点

•两种新颖的α- 1- α-呋喃糖核苷酶（ABF）显示出高的热稳定性。

•GH62家族EpABF62C的热稳定性取决于钙。

•缓冲液的pH影响两个ABF的热稳定性。

•两种ABF均可通过GH10木聚糖酶增强WAX的水解。