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Functional analysis of active site residues of Bacillus thuringiensis WB7 chitinase by site-directed mutagenesis

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Abstract

To investigate the roles of the active site residues in the catalysis of Bacillus thuringiensis WB7 chitinase, twelve mutants, F201L, F201Y, G203A, G203D, D205E, D205N, D207E, D207N, W208C, W208R, E209D and E209Q were constructed by site-directed mutagenesis. The results showed that the mutants F201L, G203D, D205N, D207E, D207N, W208C and E209D were devoid of activity, and the loss of the enzymatic activities for F201Y, G203A, D205E, W208R and E209Q were 72, 70, 48, 31 and 29%, respectively. The pH-activity profiles indicated that the optimum pH for the mutants as well as for the wildtype enzyme was 8.0. E209Q exhibited a broader active pH range while D205E, G203A and F201Y resulted in a narrower active pH range. The pH range of activity reduced 1 unit for D205E, and 2 units for G203A and F201Y. The temperature-activity profiles showed that the optimum temperature for other mutants as well as wildtype enzyme was 60°C, but 50°C for G203A, which suggested that G203A resulted in a reduction of thermostability. The study indicated that the six active site residues involving in mutagenesis played an important part in WB7 chitinase. In addition, the catalytic mechanisms of the six active site residues in WB7 chitinase were discussed.

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Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (30571257) and the Natural Science Foundation of Fujian Province (2007J0324).

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Authors and Affiliations

  1. Key Laboratory of Biopesticide and Chemical Biology, Ministry of Education, Fujian Agriculture and Forestry University, 350002, Fuzhou, Fujian, People’s Republic of China

    Wenxuan Cai, Li Sha, Jiawei Zhou, Zhipeng Huang & Xiong Guan

  2. Key Laboratory of Global Change and Marine-Atmospheric Chemistry (GCMAC), State Oceanic Administration, 361005, Xiamen, People’s Republic of China

    Wenxuan Cai

  3. Third Institute of Oceanography State Oceanic Administration, 361005, Xiamen, People’s Republic of China

    Wenxuan Cai

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  1. Wenxuan Cai
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  2. Li Sha
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Correspondence to Xiong Guan.

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Cai, W., Sha, L., Zhou, J. et al. Functional analysis of active site residues of Bacillus thuringiensis WB7 chitinase by site-directed mutagenesis. World J Microbiol Biotechnol 25, 2147–2155 (2009). https://doi.org/10.1007/s11274-009-0119-y

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