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Molecular cloning and characterization of a novel γ-CGTase from alkalophilic Bacillus sp.

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Abstract

We found a novel cyclodextrin glucanotransferase (CGTase) from alkalophilic Bacillus sp. G-825-6. The enzyme was expressed in the culture broth by recombinant Bacillus subtilis KN2 and was purified and characterized. The enzyme named CGTase825-6 showed 95% amino acid sequence identity with a known enzyme β-/γ-CGTase from Bacillus firmus/lentus 290-3. However, the product specificity of CGTase825-6 differed from that of β-/γ-CGTase. CGTase825-6 produced γ-cyclodextrin (CD) as the main product, but degradation of γ-CD was observed with prolonged reaction. The product specificity of the enzyme was positioned between γ-CGTase produced by Bacillus clarkii 7364 and B. firmus/lentus 290-3 β-/γ-CGTase. It showed that the difference of product specificity was dependent on only 28 amino acid residues in 671 residues in CGTase825-6. We compared the amino acid sequence of CGTase825-6 and those of other CGTases and constructed a protein structure model of CGTase825-6. The comparison suggested that the diminished loop (Val138-Asp142) should provide subsite -8 for γ-CD production and that Asp142 might have an important role in product specificity. CGTase825-6 should be a useful tool to produce γ-CD and to study the differences of producing mechanisms between γ-CD and β-CD.

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Authors and Affiliations

  1. Laboratory of Molecular Enzymology, Division of Life Science, Graduate School of Agricultural Science, Tohoku University, 1-1, Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai, 981 8555, Japan

    Kyoko Hirano, Takeo Ishihara, Satoshi Ogasawara, Hiroshi Maeda, Keietsu Abe, Tasuku Nakajima & Youhei Yamagata

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  1. Kyoko Hirano
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  2. Takeo Ishihara
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  3. Satoshi Ogasawara
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  4. Hiroshi Maeda
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  5. Keietsu Abe
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  6. Tasuku Nakajima
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  7. Youhei Yamagata
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Correspondence to Youhei Yamagata.

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Hirano, K., Ishihara, T., Ogasawara, S. et al. Molecular cloning and characterization of a novel γ-CGTase from alkalophilic Bacillus sp.. Appl Microbiol Biotechnol 70, 193–201 (2006). https://doi.org/10.1007/s00253-005-0041-7

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  • DOI: https://doi.org/10.1007/s00253-005-0041-7

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