Cloning, expression and biochemical characterisation of a unique thermostable pullulan-hydrolysing enzyme from the hyperthermophilic archaeon Thermococcus aggregans

Authors

  • Frank Niehaus,

    1. Institute of Technical Microbiology, Technical University Hamburg-Harburg, Denickestr. 15, 21073 Hamburg, Germany
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    • 1Brain-Biotech GmbH, Darmstaedter Strasse 34, 64673 Zwingenberg, Germany.

  • Anke Peters,

    1. Institute of Technical Microbiology, Technical University Hamburg-Harburg, Denickestr. 15, 21073 Hamburg, Germany
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  • Tatiana Groudieva,

    1. Institute of Technical Microbiology, Technical University Hamburg-Harburg, Denickestr. 15, 21073 Hamburg, Germany
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  • Garabed Antranikian

    Corresponding author
    1. Institute of Technical Microbiology, Technical University Hamburg-Harburg, Denickestr. 15, 21073 Hamburg, Germany
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*Corresponding author. Tel.: +49 (40) 42878 3117; Fax: +49 (40) 42878 2582, E-mail address: antranikian@tu-harburg.de

Abstract

The gene for a new type of pullulan hydrolase from the hyperthermophilic archaeon Thermococcus aggregans was cloned and expressed in Escherichia coli. The 2181-bp open reading frame encodes a protein of 727 amino acids. A hypothetical membrane linker region was found to be cleaved during processing in E. coli. The recombinant enzyme was purified 70-fold by heat treatment, affinity and anion exchange chromatography. Optimal activity was detected at 95°C at a broad pH range from 3.5 to 8.5 with an optimum at pH 6.5. More than 35% of enzymatic activity was detected even at 120°C. The enzyme was stable at 90°C for several hours and exhibited a half-life of 2.5 h at 100°C. Unlike all pullulan-hydrolysing enzymes described to date, the enzyme is able to attack α-1,6- as well as α-1,4-glycosidic linkages in pullulan leading to the formation of a mixture of maltotriose, panose, maltose and glucose. The enzyme is also able to degrade starch, amylose and amylopectin forming maltotriose and maltose as main products.

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