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Open Access Highly Accessed Research article

Domain-swapping of mesophilic xylanase with hyper-thermophilic glucanase

Liangwei Liu1*, Linmin Wang1, Zhang Zhang1, Xiaodan Guo1, Xiangqian Li2 and Hongge Chen13

Author Affiliations

1 Life Science College, Henan Agricultural University, 95 Wenhua Road, Zhengzhou, Henan, 450002, China

2 School of Life Science, Huaiyin Institute of Technology, 3 Meicheng Road, Huaian, Jiangsu, 223001, China

3 Key Laboratory of Enzyme Engineering of Agricultural Microbiology, 95 Wenhua Road, Zhengzhou, Henan, 450002, China

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BMC Biotechnology 2012, 12:28  doi:10.1186/1472-6750-12-28

Published: 7 June 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