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Effect of Linker Length and Flexibility on the Clostridium thermocellum Esterase Displayed on Bacillus subtilis Spores

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Abstract

In fusion protein design strategies, the flexibility and length of linkers are important parameters affecting the bioactivity of multifunctional proteins. A series of fusion proteins with different linkers were constructed. The effect of temperature, pH, and organic solvents was investigated on the enzymatic activity. Fusion proteins with P1(PTPTPT) and P2((PTPTPT)2) linkers remained highly active with wide temperature range. At pH 9.6, the relative activity of fusion proteins with (PTPTPT)2 and S2(EGKSSGSGSESKST) linkers was 70 and 62 % (1.75 and 1.5 times of that of non-linker ones). Fusion proteins with S3((GGGGS)4) linker retained 55 % activity after 5 h of incubation at 80 °C (1.2-fold of that of non-linker fusion proteins and 1.9-fold of GGGGS-linker fusion proteins). Finally, the relative activity of fusion proteins having different linkers was increased with 20 % dimethyl sulfoxide (DMSO) and methanol; relative activity of fusion proteins with EGKSSGSGSESKST linkers was enhanced 1.5- and 2.2-fold, respectively. These results suggest that longer flexible linker can enhance the activity and stability of displayed esterase than shorter flexible linker. Optimizing peptide linkers with length, flexibility, and amino acid composition could improve the thermostability and activity of the displayed enzyme.

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Acknowledgments

This study was supported by Open Funding Project of the State Key Laboratory of Bioreactor Engineering and the National Key Basic Research Program of China (973 Program, No. 2011CBA00800).

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Authors and Affiliations

  1. Institute of life sciences, Jiangsu University, Xuefu Road No.301, Zhenjiang, 212013, People’s Republic of China

    Huayou Chen, Bangguo Wu, Tianxi Zhang, Jinru Jia, Jian Lu, Zhi Chen & Zhong Ni

  2. State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, 200237, People’s Republic of China

    Huayou Chen

  3. Beijing Bioprocess Key Laboratory, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, 100029, People’s Republic of China

    Tianwei Tan

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  1. Huayou Chen
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Correspondence to Huayou Chen.

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Huayou Chen, Bangguo Wu, and Tianxi Zhang contributed equally to this work and should be considered co-first authors

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Chen, H., Wu, B., Zhang, T. et al. Effect of Linker Length and Flexibility on the Clostridium thermocellum Esterase Displayed on Bacillus subtilis Spores. Appl Biochem Biotechnol 182, 168–180 (2017). https://doi.org/10.1007/s12010-016-2318-y

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