Sandwich fusion of CBM9_2 to enhance xylanase thermostability and activity
Sandwich fusion of CBM9_2 to enhance xylanase thermostability and activity
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CBM9_2 夹心融合增强木聚糖酶热稳定性和活性
DOI：
10.1016/j.ijbiomac.2018.05.199
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发表时间：
2018
影响因子：
8.2
通讯作者：
Liu Liangwei
中科院分区：
文献类型：
--
作者：
Yang Ang;Cheng Jinsheng;Liu Meng;Shangguan Yunjie;Liu Liangwei
以嗜温黑曲霉GH 11木聚糖酶（Xyn）和嗜热海栖热袍菌GH 10木聚糖酶糖结合模块CBM9_2（C2）为夹心融合模型，将其融合到C2-Xyn-C2中。以pET 20 b-Xyn-C2为模板，用4.3kb的C2-pET 20 b大引物扩增线性化质粒C2-pET 20 b-C2-Xyn，用平末端连接法连接成环状质粒，转化E. coliBL 21（DE 3）细胞。C2-Xyn-C2在45 °C和pH 4.2下具有最佳活性，在80 °C下热灭活半衰期为2.85小时，在50 °C下热灭活半衰期为8.69小时，其中8.69小时值比Xyn和单末端融合酶Xyn-C2和C2-Xyn长24.8倍、7.5倍和7.1倍。热力学表明，该酶具有比Xyn高1.8 °C的熔融温度，较低的ΔS，ΔΔG值和更致密的结构。动力学研究表明，C2-Xyn-C2酶对山毛榉和燕麦木聚糖的催化效率分别是Xyn、Xyn-C2和C2-Xyn酶的1.2 ~6倍和2.7-~7.9倍。夹心融合使木聚糖酶进化出“盔甲手”，同时提高了热稳定性和质量活性。
Used as model for sandwich fusion, a mesophilicAspergillus nigerGH11 xylanase (Xyn) was fused into C2-Xyn-C2 with a thermophilicThermotaga maritimaGH10 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 intoE. coliBL21 (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.