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BMC Biotechnol. 2012; 12: 28.
Published online Jun 7, 2012. doi:  10.1186/1472-6750-12-28
PMCID: PMC3413519
Domain-swapping of mesophilic xylanase with hyper-thermophilic glucanase
Liangwei Liu,corresponding author1 Linmin Wang,1 Zhang Zhang,1 Xiaodan Guo,1 Xiangqian Li,2 and Hongge Chen1,3
1Life Science College, Henan Agricultural University, 95 Wenhua Road, Zhengzhou, Henan, 450002, China
2School of Life Science, Huaiyin Institute of Technology, 3 Meicheng Road, Huaian, Jiangsu, 223001, China
3Key Laboratory of Enzyme Engineering of Agricultural Microbiology, 95 Wenhua Road, Zhengzhou, Henan, 450002, China
corresponding authorCorresponding author.
Liangwei Liu: LLW321/at/yahoo.com.cn; Linmin Wang: wlm420332011/at/163.com; Zhang Zhang: heyar/at/sohu.com; Xiaodan Guo: guoxiaodan1986/at/163.com; Xiangqian Li: xiangqianli2002/at/163.com; Hongge Chen: honggeyz/at/126.com
Received January 2, 2012; Accepted June 7, 2012.
Abstract
Background
Domain fusion is limited at enzyme one terminus. The issue was explored by swapping a mesophilic Aspergillus niger GH11 xylanase (Xyn) with a hyper-thermophilic Thermotoga maritima glucanase (Glu) to construct two chimeras, Xyn-Glu and Glu-Xyn, with an intention to create thermostable xylanase containing glucanase activity.
Results
When expressed in E. coli BL21(DE3), the two chimeras exhibited bi-functional activities of xylanase and glucanase. The Xyn-Glu Xyn moiety had optimal reaction temperature (Topt) at 50 °C and thermal in-activation half-life (t1/2) at 50 °C for 47.6 min, compared to 47 °C and 17.6 min for the Xyn. The Glu-Xyn Xyn moiety had equivalent Topt to and shorter t1/2 (5.2 min) than the Xyn. Both chimera Glu moieties were more thermostable than the Glu, and the three enzyme Topt values were higher than 96 °C. The Glu-Xyn Glu moiety optimal pH was 5.8, compared to 3.8 for the Xyn-Glu Glu moiety and the Glu. Both chimera two moieties cooperated with each other in degrading substrates.
Conclusions
Domain-swapping created different effects on each moiety properties. Fusing the Glu domain at C-terminus increased the xylanase thermostability, but fusing the Glu domain at N-terminus decreased the xylanase thermostability. Fusing the Xyn domain at either terminus increased the glucanase thermostability, and fusing the Xyn domain at C-terminus shifted the glucanase pH property 2 units higher towards alkaline environments. Fusing a domain at C-terminus contributes more to enzyme catalytic activity; whereas, fusing a bigger domain at N-terminus disturbs enzyme substrate binding affinity.
Keywords: Xylanase, Glucanase, Domain-swapping, Fusing
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