title: 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.
authors.affiliation: Laboratoire de Valorisation de la Biomasse et Production des Protéines chez les Eucaryotes, Centre de Biotechnologie de Sfax, Route Sidi Mansour Km 6, PO Box 1177, 3018 Sfax, Tunisia; University of Sfax, Tunisia.
authors.forename: Naourez
authors.initials: N
authors.lastname: Damak
authors.forename: Salma
authors.initials: S
authors.lastname: Abdeljalil
authors.forename: Aida
authors.initials: A
authors.lastname: Koubaa
authors.forename: Sameh
authors.initials: S
authors.lastname: Trigui
authors.forename: Malika
authors.initials: M
authors.lastname: Ayadi
authors.forename: Hèla
authors.initials: H
authors.lastname: Trigui-Lahiani
authors.forename: Emna
authors.initials: E
authors.lastname: Kallel
authors.forename: Nadia
authors.initials: N
authors.lastname: Turki
authors.forename: Lamia
authors.initials: L
authors.lastname: Djemal
authors.forename: Hafeth
authors.initials: H
authors.lastname: Belghith
authors.forename: Noomen Hadj
authors.initials: NH
authors.lastname: Taieb
authors.forename: Ali
authors.initials: A
authors.lastname: Gargouri
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D006031:Glycosylation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010407:Penicillium / Q000201:enzymology* / Q000235:genetics*; D011133:Polysaccharide-Lyases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D059748:Proteolysis; D016415:Sequence Alignment; D013696:Temperature
chemical_list: D018076:DNA, Complementary; D011133:Polysaccharide-Lyases; C023911:pectate lyase
keywords: Heterologous expression; Medium composition; Pectate lyase