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

Applied Microbiology and Biotechnology
Dorra Zouari AyadiSamir Bejar

Abstract

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 alpha-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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Citations

Jul 30, 2014·Extremophiles : Life Under Extreme Conditions·Farah QouraGarabed Antranikian
Sep 10, 2015·Extremophiles : Life Under Extreme Conditions·Sarah RajaeiHossein Shahbani Zahiri
Mar 24, 2016·Applied Microbiology and Biotechnology·Ummirul Mukminin KaharKian Mau Goh
Nov 20, 2016·Biotechnology Letters·Shaoqing YangZhengqiang Jiang
Jun 21, 2021·Biotechnology Advances·Wei XiaJing Wu
Aug 7, 2019·Journal of Agricultural and Food Chemistry·Bo PangZhemin Zhou

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