Sandwich fusion of CBM9_2 to enhance xylanase thermostability and activity

• Published in: International journal of biological macromolecules 2018-10, Vol.117, p.586-591
• Main Authors: Yang, Ang, Cheng, Jinsheng, Liu, Meng, Shangguan, Yunjie, Liu, Liangwei
• Format: Article
• Language: English
• Subjects: Activity; Sandwich fusion; Thermostability; Xylanase
• ISSN: 0141-8130; 1879-0003
• DOI: 10.1016/j.ijbiomac.2018.05.199

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.