Molecular and biochemical characterization of <Emphasis Type="Italic">Ba</Emphasis>-EGA,a cellulase secreted by <Emphasis Type="Italic">Bacillus</Emphasis> sp. AC-1 from <Emphasis Type="Italic">Ampullaria crosseans</Emphasis> |
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Authors: | Shuang Zhang Qiu-yu Yin Yan-hong Li Ming Ding Gen-jun Xu Fu-kun Zhao |
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Institution: | (1) College of Life Science, Zhejiang Sci-Tech University, Hangzhou, 310018, China;(2) Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Graduate School of the Chinese Academy of Sciences, Shanghai, 200031, China |
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Abstract: | A novel gene (Ba-ega) of Bacillus sp. AC-1, encoding an endoglucanase (Ba-EGA), was cloned and expressed in Escherichia coli. Ba-ega, containing a 1,980-bp open reading frame (ORF), encoded a protein of 659 amino acids and had a molecular mass of 74.87 kDa.
Ba-EGA was a modular enzyme composed of a family-9 glycosyl hydrolase catalytic module (CM9) and a family-3 carbohydrate-binding
module (CBM3). To investigate the functions of the CBM3 and CM9, a number of truncated derivatives of Ba-EGA were constructed, and all were active. The catalytic module (rCM9) alone was less stable at high temperature than the
recombinant Ba-EGA (rBa-EGA). The temperature stability for the complex of rCM9 and rCBM3 was still lower than rBa-EGA, but higher than rCM9 alone. These observations indicated the existence of a non-covalent interaction between CM9 and
CBM3 that might strengthen the stability of CM9. However, this interaction is not strong enough to mimic the protective effect
of the CBM in the wild-type enzyme. |
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Keywords: | Gene cloning Expression Carbohydrate binding module Non-covalent interactions |
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