Long Liangkun, Sun Lu, Lin Qunying, Ding Shaojun, St John Franz J
College of Chemical Engineering, Nanjing Forestry University, Nanjing, 210037, China.
Institute for Microbial and Biochemical Technology, Forest Products Laboratory, USDA Forest Service, One Gifford Pinchot Drive, Madison, WI, 53726, USA.
Appl Microbiol Biotechnol. 2020 Oct;104(20):8719-8733. doi: 10.1007/s00253-020-10867-7. Epub 2020 Sep 3.
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. H 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.
已知木聚糖上的阿拉伯呋喃糖取代会干扰这种主要半纤维素的酶促水解。在本研究中，对两种新型α-L-阿拉伯呋喃糖苷酶（ABF）进行了表征和功能分析，分别是来自地霉属的TtABF51A和来自微小青霉的EpABF62C。序列分析表明，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-L-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）具有高活性，而EpABF62C更倾向于降解阿拉伯木聚糖。对于EpABF62C，RAX的水解速率（94.10 U/mg）是WAX（45.46 U/mg）的2.1倍。这两种酶的最佳pH和反应温度分别在4.0至4.5和65℃之间。钙对EpABF62C的热稳定性起重要作用。TtABF51A和EpABF62C分别在pH 4.5或5.0时表现出最高的热稳定性。在其最佳pH值下，TtABF51A和EpABF62C在55℃孵育96小时或144小时后，分别保留了大于80%的初始活性。1H NMR分析表明，这两种酶选择性地去除了与WAX中单取代木糖残基的C-3相连的阿拉伯糖。与来自微小青霉的GH10木聚糖酶EpXYN1单独应用相比，包含TtABF51A和/或EpABF62C的WAX共消化分别释放出木糖的2.49倍、3.38倍和4.81倍或木二糖的3.38倍、1.65倍和2.57倍。同时，TtABF51A与EpXYN1共同作用从WAX中释放的阿拉伯糖量是单独使用TtABF51A时的2.11倍。要点：• 两种新型α-L-阿拉伯呋喃糖苷酶（ABF）表现出高热稳定性。• GH62家族的EpABF62C的热稳定性依赖于钙。• 缓冲液pH影响这两种ABF的热稳定性。• 两种ABF均增强了GH10木聚糖酶对WAX的水解作用。
