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Cloning and heterologous expression of a thermostable pectate lyase from Penicillium occitanis in Escherichia coli.

Abstract
The entire pectate lyase cDNA (Pel1) of Penicillium occitanis was cloned from a cDNA bank and sequenced. The ORF exhibited a great homology to Penicillium marneffei and conservation of all features of fungal pectate lyases such as the barrel structure with "eight right-handed parallel β-helix" architecture. The structure modeling also showed the interesting resemblance with thermostable pectate lyases since several specific residues were also shared by Pel1 and these thermostable enzymes. Having shown that the enzyme retains its activity after endoH-mediated deglycosylation, we investigated its expression in Escherichia coli BL21 using the pET28-a vector. This expression was shown to be optimum when cells were induced at room temperature in 2YT medium rather than at 37 °C and LB medium. In such conditions, the recombinant protein was apparently produced more in soluble form than as inclusion bodies. The effect of NaCl concentration was investigated during the binding and elution steps of recombinant His-tagged enzyme on MagneHis Ni-particles. The purified enzyme was shown to retain its thermo-activity as well as a great tolerance to high concentration of NaCl and imidazole.
AuthorsNaourez Damak, Salma Abdeljalil, Aida Koubaa, Sameh Trigui, Malika Ayadi, Hèla Trigui-Lahiani, Emna Kallel, Nadia Turki, Lamia Djemal, Hafeth Belghith, Noomen Hadj Taieb, Ali Gargouri
JournalInternational journal of biological macromolecules (Int J Biol Macromol) Vol. 62 Pg. 549-56 (Nov 2013) ISSN: 1879-0003 [Electronic] Netherlands
PMID24141072 (Publication Type: Journal Article)
CopyrightCopyright © 2013 Elsevier B.V. All rights reserved.
Chemical References
  • DNA, Complementary
  • Polysaccharide-Lyases
  • pectate lyase
Topics
  • Amino Acid Sequence
  • Base Sequence
  • Cloning, Molecular
  • DNA, Complementary (chemistry, genetics, metabolism)
  • Enzyme Activation
  • Enzyme Stability
  • Escherichia coli (genetics, metabolism)
  • Gene Expression
  • Glycosylation
  • Models, Molecular
  • Molecular Sequence Data
  • Penicillium (enzymology, genetics)
  • Polysaccharide-Lyases (chemistry, genetics, isolation & purification, metabolism)
  • Protein Conformation
  • Proteolysis
  • Sequence Alignment
  • Temperature

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