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Characterisation of a thermoalkali-stable cyclodextrin glycosyltransferase from the anaerobic thermoalkaliphilic bacterium Anaerobranca gottschalkii

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Abstract

The thermoalkaliphilic anaerobic bacterium Anaerobranca gottschalkii produces an extracellular CGTase when grown on starch at 55°C and pH 9.0. The gene encoding this CGTase was cloned and successfully expressed in Escherichia coli. It encodes a protein consisting of 721 amino acids with a signal sequence of 34 amino acids. On SDS–polyacrylamide gels, the purified CGTase from A. gottschalkii displayed the expected molecular mass of 78 kDa. The recombinant enzyme was purified with a yield of 13.5% and displayed a specific activity of 210 units/mg. This CGTase, which represents the first report of a CGTase from an anaerobic thermoalkaliphile, was active at a broad range of temperature and pH, namely 55–70°C and pH 5–10. It completely converted amylose, amylopectin and native starch to cyclodextrins, preferentially α-cyclodextrin. With a longer incubation period, the α-cyclodextrin to β-cyclodextrin ratio declined. Variations in substrate type and concentration influenced the product pattern. Increasing the substrate concentration (0.5–20.0%) and glucans containing branching points (α-1,6 glycosidic linkages) shifted the product pattern to: β-cyclodextin > α-cyclodextrin > γ-cyclodextrin. In addition to these cyclodextrins, larger cyclodextrins (>8 glucose units) were formed in the initial reaction period. The CGTase was stabilised against thermal inactivation by calcium ions and high substrate concentrations; and 5 mM of CaCl2 shifted the apparent melting point of the enzyme from 60°C to 69°C.

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Acknowledgement

We thank the Deutsche Bundesstiftung Umwelt (German Federal Environmental Foundation) for financial support.

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Author notes

  1. Catharina Dönges & Reinhard Sterner

    Present address: Institute of Biophysics and Physical Biochemistry, University of Regensburg, Universitätsstrasse 31, 93053, Regensburg, Germany

Authors and Affiliations

  1. Institute of Technical Microbiology, Technical University Hamburg-Harburg, Kasernenstrasse 12, 21073, Hamburg, Germany

    Volker Thiemann, Steffen G. Prowe & Garabed Antranikian

  2. Institute of Biochemistry, University of Cologne, Otto-Fischer-Strasse 12, 50674, Cologne, Germany

    Catharina Dönges & Reinhard Sterner

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  1. Volker Thiemann
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  2. Catharina Dönges
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  5. Garabed Antranikian
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Correspondence to Garabed Antranikian.

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Dedicated to Prof. Dr. Hans G. Schlegel on the occasion of his 80th birthday.

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Thiemann, V., Dönges, C., Prowe, S.G. et al. Characterisation of a thermoalkali-stable cyclodextrin glycosyltransferase from the anaerobic thermoalkaliphilic bacterium Anaerobranca gottschalkii. Arch Microbiol 182, 226–235 (2004). https://doi.org/10.1007/s00203-004-0717-x

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  • DOI: https://doi.org/10.1007/s00203-004-0717-x

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