Applied Microbiology and Biotechnology

, Volume 70, Issue 2, pp 193–201

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

  • Kyoko Hirano
  • Takeo Ishihara
  • Satoshi Ogasawara
  • Hiroshi Maeda
  • Keietsu Abe
  • Tasuku Nakajima
  • Youhei Yamagata
Biotechnologically Relevant Enzymes and Proteins

DOI: 10.1007/s00253-005-0041-7

Cite this article as:
Hirano, K., Ishihara, T., Ogasawara, S. et al. Appl Microbiol Biotechnol (2006) 70: 193. doi:10.1007/s00253-005-0041-7

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.

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Kyoko Hirano
    • 1
  • Takeo Ishihara
    • 1
  • Satoshi Ogasawara
    • 1
  • Hiroshi Maeda
    • 1
  • Keietsu Abe
    • 1
  • Tasuku Nakajima
    • 1
  • Youhei Yamagata
    • 1
  1. 1.Laboratory of Molecular Enzymology, Division of Life Science, Graduate School of Agricultural ScienceTohoku UniversitySendaiJapan