title: Enhancement of solubility, purification and inclusion-bodies-refolding of an active pectin lyase from Penicillium occitanis expressed in Escherichia coli.
abstract: Pectin lyase (pnl) is the only pectinase able to hydrolyze directly the highly methylated pectin without liberating the toxic methanol and without disturbing ester content responsible for specific aroma of juices. The cDNA of Penicillium occitanis pnl (mature form) was cloned into pET-21a as expression vector and over-expressed into Esherichia coli. Most of recombinant pnl was expressed as inclusion bodies. Pnl activity was confirmed by colorimetric assay. To enhance the solubility yield of the expressed pnl, the effects of induction temperature, host strain and expression level were optimized. Maximal production of functional pnl was obtained after induction by 0.4mM IPTG at 30°C and 150rpm for 16h. Interestingly, the use of Origami host strain, having an oxidized cytoplasm favoring disulfide bonds formation required for the active conformation of the enzyme, has significantly improved the yield of the soluble active form of recombinant pnl. This pnl was successfully purified through a single step purification using His-Trap affinity column chromatography. This work is the first to report pnl expression into Origami strain. Alternatively, the inclusion bodies were isolated, denatured by high concentration of urea and gradually refolded by successive dialysis, leading to their transformation into soluble and active form.
authors.affiliation: Laboratoire de Biotechnologie Moléculaire des Eucaryotes, Centre de Biotechnologie de Sfax, PB1177, University of Sfax, Tunisia.
authors.forename: Azza
authors.initials: A
authors.lastname: Hadj Sassi
authors.affiliation: Laboratoire de Biotechnologie Moléculaire des Eucaryotes, Centre de Biotechnologie de Sfax, PB1177, University of Sfax, Tunisia.
authors.forename: Hèla
authors.initials: H
authors.lastname: Trigui-Lahiani
authors.affiliation: Laboratoire de Biotechnologie Moléculaire des Eucaryotes, Centre de Biotechnologie de Sfax, PB1177, University of Sfax, Tunisia.
authors.forename: Salma
authors.initials: S
authors.lastname: Abdeljalil
authors.affiliation: Laboratoire de Biotechnologie Moléculaire des Eucaryotes, Centre de Biotechnologie de Sfax, PB1177, University of Sfax, Tunisia. Electronic address: Faouzi.gargouri@cbs.rnrt.tn.
authors.forename: Ali
authors.initials: A
authors.lastname: Gargouri
mesh_terms: D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D002479:Inclusion Bodies / Q000201:enzymology*; D010407:Penicillium / Q000201:enzymology* / Q000235:genetics; D011133:Polysaccharide-Lyases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D058849:Protein Refolding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D012995:Solubility; D013696:Temperature
chemical_list: D011994:Recombinant Proteins; D011133:Polysaccharide-Lyases; C027349:pectin lyase
keywords: Heterologous expression; Inclusion bodies; Pectin lyase