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Mutation of Tyrosine167Histidine at Remote Substrate Binding Subsite −6 in α-Cyclodextrin Glycosyltransferase Enhancing α-Cyclodextrin Specificity by Directed Evolution

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Abstract

α-Cyclodextrin glycosyltransferase (α-CGTase) can convert starch into α-cyclodextrin with various proportions of β-cyclodextrin and/or γ-cyclodextrin in the products. To improve the α-cyclodextrin-forming specificity, directed evolution on the wild-type α-CGTase was performed by constructing mutant library with error-prone PCR method. The positive mutant strains were selected in combination of starch plate screening with HPLC detection of the products. An α-CGTase from the mutant strain (assigned No. 95) was found to be able to increase the α:β ratio in product mixture from 3.4 to 7.8 in comparison with the wild-type α-CGTase. Sequence alignment indicated that two mutations occurred in the No. 95 mutant α-CGTase, which were Y167H and A536V. Reverse mutation revealed that Y167H was responsible for this change. A series of 167 site-substituted mutants could improve the α:β ratio to different extents as indicated by saturated mutagenesis, with Y167H as the best substitution. In conclusion, Y167 was confirmed to be one of the main subsites in the −6 domain of α-CGTase that is responsible for the α:β ratio in the product mixture. Y167H is most preferable among all types of mutant enzymes tested at this site. The reconstructed Y167H (i.e., No. 95) α-CGTase showed better potential for α-cyclodextrin production on industrial scale.

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Acknowledgments

This work was supported by the National Natural Science Foundation of People’s Republic of China (Project No. 31171643).

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Authors and Affiliations

  1. State Key Laboratories of Transducer Technology, National Engineering Lab for Industrial Enzymes, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China

    Binghong Song, Yang Yue, Ting Xie, Shijun Qian & Yapeng Chao

  2. University of Chinese Academy of Sciences, Beijing, 100101, China

    Ting Xie

  3. School of Biological Science and Medical Engineering, BUAA, Beijing, 100101, China

    Yang Yue

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  1. Binghong Song
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  2. Yang Yue
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Correspondence to Yapeng Chao.

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Song, B., Yue, Y., Xie, T. et al. Mutation of Tyrosine167Histidine at Remote Substrate Binding Subsite −6 in α-Cyclodextrin Glycosyltransferase Enhancing α-Cyclodextrin Specificity by Directed Evolution. Mol Biotechnol 56, 232–239 (2014). https://doi.org/10.1007/s12033-013-9699-8

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