C-Terminal carbohydrate-binding module 9_2 fused to the N-terminus of GH11 xylanase from Aspergillus niger.
OBJECTIVE
The 9_2 carbohydrate-binding module (C2) locates natively at the C-terminus of the GH10 thermophilic xylanase from Thermotoga marimita. When fused to the C-terminus, C2 improved thermostability of a GH11 xylanase (Xyn) from Aspergillus niger. However, a question is whether the C-terminal C2 would have a thermostabilizing effect when fused to the N-terminus of a catalytic module.


RESULTS
A chimeric enzyme, C2-Xyn, was created by step-extension PCR, cloned in pET21a(+), and expressed in E. coli BL21(DE3). The C2-Xyn exhibited a 2 °C higher optimal temperature, a 2.8-fold longer thermostability, and a 4.5-fold higher catalytic efficiency on beechwood xylan than the Xyn. The C2-Xyn exhibited a similar affinity for binding to beechwood xylan and a higher affinity for oat-spelt xylan than Xyn.


CONCLUSIONS
C2 is a thermostabilizing carbohydrate-binding module and provides a model of fusion at an enzymatic terminus inconsistent with the modular natural terminal location.
From the Life Science College, Henan Agricultural University, 95 Wenhua Road, Zhengzhou, 450002, Henan, People's Republic of China.
Wenxuan
W
Xu
From the Life Science College, Henan Agricultural University, 95 Wenhua Road, Zhengzhou, 450002, Henan, People's Republic of China.
Yajuan
Y
Liu
From the Life Science College, Henan Agricultural University, 95 Wenhua Road, Zhengzhou, 450002, Henan, People's Republic of China.
Yanxin
Y
Ye
From the Life Science College, Henan Agricultural University, 95 Wenhua Road, Zhengzhou, 450002, Henan, People's Republic of China.
Meng
M
Liu
From the Life Science College, Henan Agricultural University, 95 Wenhua Road, Zhengzhou, 450002, Henan, People's Republic of China.
Laichuang
L
Han
From the Life Science College, Henan Agricultural University, 95 Wenhua Road, Zhengzhou, 450002, Henan, People's Republic of China.
Andong
A
Song
From the Life Science College, Henan Agricultural University, 95 Wenhua Road, Zhengzhou, 450002, Henan, People's Republic of China. liangwei@henau.edu.cn.|Key Laboratory of Enzyme Engineering of Agricultural Microbiology, Ministry of Agriculture, Zhengzhou, People's Republic of China. liangwei@henau.edu.cn.
Liangwei
L
Liu
D001234:Aspergillus niger / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D020134:Catalytic Domain; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D020124:Thermotoga maritima / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D043364:Endo-1,4-beta Xylanases
Aspergillus niger; Carbohydrate-binding module 9_2; N-terminal fusion; Xylanase