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Heterologous expression and characterization of a novel branching enzyme from the thermoalkaliphilic anaerobic bacterium Anaerobranca gottschalkii

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Abstract

The gene encoding the branching enzyme (BE) from the thermoalkaliphilic, anaerobic bacterium Anaerobranca gottschalkii was fused with a twin arginine translocation protein secretory-pathway-dependent signal sequence from Escherichia coli and expressed in Staphylococcus carnosus. The secreted BE was purified using hydrophobic interaction and gel filtration chromatography. The monomeric enzyme (72 kDa) shows maximal activity at 50°C and pH 7.0. With amylose the BE displays high transglycosylation and extremely low hydrolytic activity. The conversion of amylose and linear dextrins was analysed by applying high-performance anion exchange chromatography and quantitative size-exclusion chromatography. Amylose (104–4×107 g/mol) was converted to a major extent to products displaying molecular masses of 104–4×105 g/mol, indicating that the enzyme could be applicable for the production of starch or dextrins with narrow molecular mass distributions. The majority of the transferred oligosaccharides, determined after enzymatic hydrolysis of the newly synthesized α-1,6 linkages, ranged between 103 and 104 g/mol, which corresponds to a degree of polymerisation (DP) of 6–60. The minimal donor chain length is DP 16. Furthermore, the obtained results support the hypotheses of a random endocleavage mechanism of BE and the occurrence of interchain branching.

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Acknowledgements

The authors thank Martina Reisen and Sascha Lebioda for their valuable technical contribution and the Deutsche Bundesstiftung Umwelt (German Federal Environmental Foundation) for financial support.

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Authors and Affiliations

  1. Institute of Technical Microbiology, Hamburg University of Technology, 21073, Hamburg, Germany

    Volker Thiemann, Costanzo Bertoldo & Garabed Antranikian

  2. Institute for Wood Chemistry and Chemical Technology of Wood, Federal Research Centre for Forestry and Forest Products, 21002, Hamburg, Germany

    Bodo Saake & Jürgen Puls

  3. Biotechnology I, Research Centre Jülich GmbH, 52425, Jülich, Germany

    Angela Vollstedt & Roland Freudl

  4. Microbial Discovery and Molecular Biotechnology, Novozymes, 2880, Bagsvaerd, Denmark

    Thomas Schäfer

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  1. Volker Thiemann
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Correspondence to Garabed Antranikian.

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Thiemann, V., Saake, B., Vollstedt, A. et al. Heterologous expression and characterization of a novel branching enzyme from the thermoalkaliphilic anaerobic bacterium Anaerobranca gottschalkii . Appl Microbiol Biotechnol 72, 60–71 (2006). https://doi.org/10.1007/s00253-005-0248-7

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