State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu 214122, China.
School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu 214122, China.
Biotechnol Biofuels. 2014 Jan 6;7(1):3. doi: 10.1186/1754-6834-7-3.
Xylanases have drawn much attention owing to possessing great potential in various industrial applications. However, the applicability of xylanases, exemplified by the production of bioethanol and xylooligosaccharides (XOSs), was bottlenecked by their low stabilities at higher temperatures. The main purpose of this work was to improve the thermostability of AuXyn11A, a mesophilic glycoside hydrolase (GH) family 11 xylanase from Aspergillus usamii E001, by N-terminus replacement.
A hybrid xylanase with high thermostability, named AEXynM, was predicted by computational methods, and constructed by substituting the N-terminal 33 amino acids of AuXyn11A with the corresponding 38 ones of EvXyn11TS, a hyperthermostable family 11 xylanase. Two AuXyn11A- and AEXynM-encoding genes, Auxyn11A and AExynM, were then highly expressed in Pichia pastoris GS115, respectively. The specific activities of two recombinant xylanases (reAuXyn11A and reAEXynM) were 10,437 and 9,529 U mg-1. The temperature optimum and stability of reAEXynM reached 70 and 75°C, respectively, much higher than those (50 and 45°C) of reAuXyn11A. The melting temperature (Tm) of reAEXynM, measured using the Protein Thermal Shift (PTS) method, increased by 34.0°C as compared with that of reAuXyn11A. Analyzed by HPLC, xylobiose and xylotriose as the major hydrolytic products were excised from corncob xylan by reAEXynM. Additionally, three single mutant genes from AExynM (AExynMC5T, AExynMP9S, and AExynMH14N) were constructed by site-directed mutagenesis as designed theoretically, and expressed in P. pastoris GS115, respectively. The thermostabilities of three recombinant mutants clearly decreased as compared with that of reAEXynM, which demonstrated that the three amino acids (Cys5, Pro9, and His14) in the replaced N-terminus contributed mainly to the high thermostability of AEXynM.
This work highly enhanced the thermostability of AuXyn11A by N-terminus replacement, and further verified, by site-directed mutagenesis, that Cys5, Pro9, and His14 contributed mainly to the improved thermostability. It will provide an effective strategy for improving the thermostabilities of other enzymes.
木聚糖酶由于在各种工业应用中具有巨大的潜力而备受关注。然而，木聚糖酶的适用性，例如生物乙醇和木低聚糖（XOS）的生产，受到其在较高温度下低稳定性的限制。这项工作的主要目的是通过 N 端替换来提高 Aspergillus usamii E001 中嗜温糖苷水解酶（GH）家族 11 木聚糖酶 AuXyn11A 的热稳定性。
通过计算方法预测了一种具有高热稳定性的杂合木聚糖酶，命名为 AEXynM，并通过用对应于 EvXyn11TS 的 38 个氨基酸替换 AuXyn11A 的 N 端 33 个氨基酸来构建，EvXyn11TS 是一种超耐热家族 11 木聚糖酶。然后，分别在毕赤酵母 GS115 中高度表达了两个编码 AuXyn11A 和 AEXynM 的基因 Auxyn11A 和 AExynM。两个重组木聚糖酶（reAuXyn11A 和 reAEXynM）的比活性分别为 10437 和 9529 U mg-1。reAEXynM 的最适温度和稳定性分别达到 70°C 和 75°C，明显高于 reAuXyn11A（分别为 50°C 和 45°C）。使用蛋白质热移位（PTS）方法测量的 reAEXynM 的熔点（Tm）比 reAuXyn11A 升高了 34.0°C。通过 HPLC 分析，reAEXynM 从玉米芯木聚糖中切除了木二糖和木三糖作为主要水解产物。此外，通过定点突变理论设计，分别构建了来自 AExynM 的三个单突变基因（AExynMC5T、AExynMP9S 和 AExynMH14N），并分别在毕赤酵母 GS115 中表达。与 reAEXynM 相比，三个重组突变体的热稳定性明显降低，这表明替换的 N 端的三个氨基酸（半胱氨酸 5、脯氨酸 9 和组氨酸 14）主要有助于提高 AEXynM 的热稳定性。
本工作通过 N 端替换显著提高了 AuXyn11A 的热稳定性，并通过定点突变进一步验证了 Cys5、Pro9 和 His14 主要有助于提高热稳定性。它将为提高其他酶的热稳定性提供一种有效的策略。
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