the Life Science College, Henan Agricultural University, Zhengzhou 450002, China.
the Life Science College, Henan Agricultural University, Zhengzhou 450002, China; The Key Laboratory of Enzyme Engineering of Agricultural Microbiology, Ministry of Agriculture, Henan Agricultural University, Zhengzhou 450002, China.
Int J Biol Macromol. 2018 Oct 1;117:586-591. doi: 10.1016/j.ijbiomac.2018.05.199. Epub 2018 May 28.
Used as model for sandwich fusion, a mesophilic Aspergillus niger GH11 xylanase (Xyn) was fused into C2-Xyn-C2 with a thermophilic Thermotaga maritima GH10 xylanase carbohydrate-binding module CBM9_2 (C2). Linearized plasmids C2-pET20b-C2-Xyn were amplified from template pET20b-Xyn-C2 with a 4.3 kb C2-pET20b megaprimer, ligated into circular plasmids in blunt-end ligation, and transformed into E. coli BL21 (DE3) cells. The C2-Xyn-C2 had optimum activity at 45 °C and pH 4.2, a 2.85 h thermal inactivation half-life at 80 °C and a 8.69 h at 50 °C, with the 8.69 h value 24.8-, 7.5-, and 7.1-fold longer than the Xyn and single terminal fusion enzymes Xyn-C2, and C2-Xyn. Thermodynamics showed that the enzyme had a 1.8 °C higher melting temperature, lower values ΔS, ΔΔG, and a denser structure than the Xyn. Kinetics showed that the C2-Xyn-C2 catalytic efficiency was 1.2-6-fold and 2.7-7.9-fold higher on beechwood and oat-spelt xylan than those of the enzymes Xyn, Xyn-C2, and C2-Xyn. The sandwich fusion evolved the xylanase with "armor-hands" to enhance simultaneously thermostability and activity in quality.
作为三明治融合的模型，嗜温黑曲霉 GH11 木聚糖酶（Xyn）与嗜热海洋栖热菌 GH10 木聚糖酶碳水化合物结合模块 CBM9_2（C2）融合成 C2-Xyn-C2。线性化质粒 C2-pET20b-C2-Xyn 从模板 pET20b-Xyn-C2 用 4.3 kb C2-pET20b 大片段扩增，在平头连接中连接成环状质粒，并转化到大肠杆菌 BL21（DE3）细胞中。C2-Xyn-C2 在 45°C 和 pH 4.2 下具有最佳活性，在 80°C 下的热失活动力学半衰期为 2.85 小时，在 50°C 下为 8.69 小时，8.69 小时的值比 Xyn 和单末端融合酶 Xyn-C2 和 C2-Xyn 长 24.8、7.5 和 7.1 倍。热力学表明，与 Xyn 相比，该酶的熔点高 1.8°C，ΔS、ΔΔG 值更低，结构更密集。动力学表明，C2-Xyn-C2 对桦木和燕麦-spelt 木聚糖的催化效率比 Xyn、Xyn-C2 和 C2-Xyn 高 1.2-6 倍和 2.7-7.9 倍。三明治融合使木聚糖酶进化出“装甲手”，以同时提高热稳定性和活性。
