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Heterologous expression, secretion and characterization of the Geobacillus thermoleovorans US105 type I pullulanase


Pullulanase type I of Geobacillus thermoleovorans US105 strain (PUL US105) was produced and secreted efficiently in the E. coli periplasmic or extracellular fraction using two different signal peptides. Hence, the open reading frame was connected downstream of the lipase A signal peptide of Bacillus subtilis strain leading to an efficient secretion of an active form enzyme on the periplasmic fraction. In addition, pul US105 was fused to the α-amylase signal sequence of the Bacillus stearothermophilus US100 strain. The monitoring of the pullulanase activity and Western blot analysis for this last construction showed that the most activity was found in the supernatant culture, proving the efficient secretion of this natively cytoplasmic enzyme as an active form. The PUL US105 was purified to homogeneity from the periplasmic fraction, using heat treatment, size exclusion, and anion-exchange chromatography. The native pullulanase has a molecular mass of 160 kDa and is composed of two identical subunits of 80 kDa each. It was independent for metallic ions for its activity, while its thermostability was obviously improved in presence of only 0.1 mM CaCl2.

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This research was supported by the Tunisian Government “Contract Programme CBS-LEMP.” Authors show gratitude to Mr. Naili, B. for their technical assistance. We thank Ms. Masmoudi, N. for her collaboration especially with the FPLC apparatus. We are also grateful to Mr. Olivera Francetic (Unité de génétique Moléculaire, Institut Pasteur, Paris) for kindly providing antipullulanase antibodies. We also thank Dr. Thorsten Eggert, Directed Evolution Group, Institute of Molecular Enzyme Technology Heinrich-Heine-University, Düsseldorf Research Centre Jülich, D-52426, Jülich, Germany, for kindly providing pBSMul2 vector to Dr. Hichem Chouayekh.

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Correspondence to Samir Bejar.

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Zouari Ayadi, D., Ben Ali, M., Jemli, S. et al. Heterologous expression, secretion and characterization of the Geobacillus thermoleovorans US105 type I pullulanase. Appl Microbiol Biotechnol 78, 473–481 (2008).

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  • Secretion
  • Signal peptide
  • Pullulanase
  • Purification