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 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.
Zouari Ayadi
Dorra
D
Laboratory of Enzymes and Metabolites of Prokaryotes, Centre of Biotechnology of Sfax, BP K, 3038, Sfax, Tunisia.
Ben Ali
Mamdouh
M
Jemli
Sonia
S
Ben Mabrouk
Sameh
S
Mezghani
Monia
M
Ben Messaoud
Ezzedine
E
Bejar
Samir
S
D001406:Bacillaceae / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002845:Chromatography; D003001:Cloning, Molecular; D004795:Enzyme Stability; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008049:Lipase / Q000737:chemistry; D008970:Molecular Weight; D015202:Protein Engineering; D021382:Protein Sorting Signals / Q000235:genetics; D021381:Protein Transport; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature; D000516:alpha-Amylases / Q000737:chemistry
D001426:Bacterial Proteins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D008049:Lipase; D006026:Glycoside Hydrolases; D000516:alpha-Amylases; C100181:pullulanase