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Functional and structural studies of pullulanase from Anoxybacillus sp. LM18-11

Authors

  • Jianyong Xu,

    1. Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China
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    • Jianyong Xu and Feifei Ren contributed equally to this work.

  • Feifei Ren,

    1. Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China
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    • Jianyong Xu and Feifei Ren contributed equally to this work.

  • Chun-Hsiang Huang,

    1. Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China
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  • Yingying Zheng,

    1. Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China
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  • Jie Zhen,

    1. Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China
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  • Hong Sun,

    1. Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China
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  • Tzu-Ping Ko,

    1. Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan
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  • Miao He,

    1. Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China
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  • Chun-Chi Chen,

    1. Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China
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  • Hsiu-Chien Chan,

    1. Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China
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  • Rey-Ting Guo,

    Corresponding author
    1. Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China
    • Correspondence to: Yanhe Ma, 32 XiQiDao, Tianjin Airport Economic Park, Tianjin 300308, China. E-mail: ma_yh@tib.cas.cn; Hui Song, 32 XiQiDao, Tianjin Airport Economic Park, Tianjin 300308, China. E-mail: song_h@tib.cas.cn; and Rey-Ting Guo, 32 XiQiDao, Tianjin Airport Economic Park, Tianjin 300308, China. E-mail: guo_rt@tib.cas.cn

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  • Hui Song,

    Corresponding author
    1. Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China
    • Correspondence to: Yanhe Ma, 32 XiQiDao, Tianjin Airport Economic Park, Tianjin 300308, China. E-mail: ma_yh@tib.cas.cn; Hui Song, 32 XiQiDao, Tianjin Airport Economic Park, Tianjin 300308, China. E-mail: song_h@tib.cas.cn; and Rey-Ting Guo, 32 XiQiDao, Tianjin Airport Economic Park, Tianjin 300308, China. E-mail: guo_rt@tib.cas.cn

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  • Yanhe Ma

    Corresponding author
    1. Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China
    • Correspondence to: Yanhe Ma, 32 XiQiDao, Tianjin Airport Economic Park, Tianjin 300308, China. E-mail: ma_yh@tib.cas.cn; Hui Song, 32 XiQiDao, Tianjin Airport Economic Park, Tianjin 300308, China. E-mail: song_h@tib.cas.cn; and Rey-Ting Guo, 32 XiQiDao, Tianjin Airport Economic Park, Tianjin 300308, China. E-mail: guo_rt@tib.cas.cn

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ABSTRACT

Pullulanase is a debranching enzyme that specifically hydrolyzes the α-1,6 glycosidic linkage of α-glucans, and has wide industrial applications. Here, we report structural and functional studies of a new thermostable pullulanase from Anoxybacillus sp. LM18-11 (PulA). Based on the hydrolysis products, PulA was classified as a type I pullulanase. It showed maximum activity at 60°C and pH 6.0. Kinetic study showed that the specific activity and Km for pullulan of PulA are 750 U mg−1 and 16.4 μmol L−1, respectively. PulA has a half-life of 48 h at 60°C. The remarkable thermostability makes PulA valuable for industrial usage. To further investigate the mechanism of the enzyme, we solved the crystal structures of PulA and its complexes with maltotriose and maltotetraose at 1.75–2.22 Å resolution. The PulA structure comprises four domains (N1, N2, A, and C). A is the catalytic domain, in which three conserved catalytic residues were identified (D413, E442, and D526). Two molecules of oligosaccharides were seen in the catalytic A domain in a parallel binding mode. Interestingly, another two oligosaccharides molecules were found between the N1 domain and the loop between the third β-strand and the third α-helix in the A domain. Based on sequence alignment and the ligand binding pattern, the N1 domain is identified as a new type of carbohydrate-binding motif and classified to the CBM68 family. The structure solved here is the first structure of pullulanase which has carbohydrate bound to the N1 domain. Proteins 2014; 82:1685–1693. © 2013 Wiley Periodicals, Inc.

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