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Applied and Environmental Microbiology, June 2004, p. 3407-3416, Vol. 70, No. 6
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.6.3407-3416.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Cloning, Sequencing, and Characterization of a Heat- and Alkali-Stable Type I Pullulanase from Anaerobranca gottschalkii

Costanzo Bertoldo,¹ Martin Armbrecht,² Fiona Becker,³ Thomas Schäfer,³ Garabed Antranikian,^1* and Wolfgang Liebl²

Technical Microbiology, Technical University of Hamburg-Harburg, D-21073 Hamburg,¹ Institute of Microbiology and Genetics, Georg August University, D-37077 Göttingen, Germany,² Molecular Biotechnology, Novozymes A/S, 2880 Bagsværd, Denmark³

Received 7 November 2003/ Accepted 6 March 2004

The gene encoding a type I pullulanase was identified from the genome sequence of the anaerobic thermoalkaliphilic bacterium Anaerobranca gottschalkii. In addition, the homologous gene was isolated from a gene library of Anaerobranca horikoshii and sequenced. The proteins encoded by these two genes showed 39% amino acid sequence identity to the pullulanases from the thermophilic anaerobic bacteria Fervidobacterium pennivorans and Thermotoga maritima. The pullulanase gene from A. gottschalkii (encoding 865 amino acids with a predicted molecular mass of 98 kDa) was cloned and expressed in Escherichia coli strain BL21(DE3) so that the protein did not have the signal peptide. Accordingly, the molecular mass of the purified recombinant pullulanase (rPulAg) was 96 kDa. Pullulan hydrolysis activity was optimal at pH 8.0 and 70°C, and under these physicochemical conditions the half-life of rPulAg was 22 h. By using an alternative expression strategy in E. coli Tuner(DE3)(pLysS), the pullulanase gene from A. gottschalkii, including its signal peptide-encoding sequence, was cloned. In this case, the purified recombinant enzyme was a truncated 70-kDa form (rPulAg'). The N-terminal sequence of purified rPulAg' was found 252 amino acids downstream from the start site, presumably indicating that there was alternative translation initiation or N-terminal protease cleavage by E. coli. Interestingly, most of the physicochemical properties of rPulAg' were identical to those of rPulAg. Both enzymes degraded pullulan via an endo-type mechanism, yielding maltotriose as the final product, and hydrolytic activity was also detected with amylopectin, starch, ß-limited dextrins, and glycogen but not with amylose. This substrate specificity is typical of type I pullulanases. rPulAg was inhibited by cyclodextrins, whereas addition of mono- or bivalent cations did not have a stimulating effect. In addition, rPulAg' was stable in the presence of 0.5% sodium dodecyl sulfate, 20% Tween, and 50% Triton X-100. The pullulanase from A. gottschalkii is the first thermoalkalistable type I pullulanase that has been described.

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* Corresponding author. Mailing address: Technical University of Hamburg-Harburg, Technical Microbiology, Kasernenstrasse 12, D-21073 Hamburg, Germany. Phone: 49-40-428783117. Fax: 49-40-428782582. E-mail: antranikian{at}tuhh.de.

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Applied and Environmental Microbiology, June 2004, p. 3407-3416, Vol. 70, No. 6
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.6.3407-3416.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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