Exploration of a N-terminal disulfide bridge to improve the thermostability of a GH11 xylanase from Aspergillus niger.
To improve the thermostability of xylanase XynZF-2 from Aspergillus niger XZ-3S, a disulfide bridge was introduced in the N-terminal domains by site-directed mutagenesis (V1C and E27C). Simultaneously, the active sites of XynZF-2 were predicted by bioinformatics software and verified by site-directed mutagenesis (E103D and E194D). The mutated active sites xynED- and the mutated disulfide bridge xynDC-encoding genes were constructed and expressed in Escherichia coli BL21 (DE3). Compared to the native xylanase, it was found that the residual activity of the mutated XynED was 0.17%. The optimum temperature of the variant XynDC was increased to 45°C from 40°C of XynZF-2. After treatment at 40°C for 60 min, the variant XynDC retained 66.77% of their original activity, while the XynZF-2 retained about 44.36% residual activity. t1/2(45°C) of the variant XynDC also increased from 7 min to 14 min. The results of the mutated xylanases indicated that the active center of XynZF-2 mainly consisted of two catalytic residues (Glu103 and Glu194), and the introduction of a disulfide bridge in the N-terminal domains can improve the thermostability of XynZF-2.
School of Life Science and Technology, Xinxiang Medical University.
Chen-Yan
CY
Zhou
Tong-Biao
TB
Li
Yong-Tao
YT
Wang
Xin-Shu
XS
Zhu
Jing
J
Kang
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001234:Aspergillus niger / Q000201:enzymology*; D020134:Catalytic Domain; D004220:Disulfides / Q000737:chemistry; D043364:Endo-1,4-beta Xylanases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D013696:Temperature
D004220:Disulfides; D043364:Endo-1,4-beta Xylanases