Enhancement of solubility, purification and inclusion-bodies-refolding of an active pectin lyase from Penicillium occitanis expressed in Escherichia coli
| Autor: | Ali Gargouri, Salma Abdeljalil, Hèla Trigui-Lahiani, Azza Hadj Sassi |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
food.ingredient Pectin Gene Expression medicine.disease_cause Biochemistry Protein Refolding Inclusion bodies 03 medical and health sciences food Affinity chromatography Structural Biology Escherichia coli medicine Cloning Molecular Pectinase Molecular Biology Polysaccharide-Lyases Pectin lyase Inclusion Bodies Chromatography Chemistry Penicillium Temperature Sequence Analysis DNA General Medicine Penicillium occitanis Recombinant Proteins 030104 developmental biology Solubility Heterologous expression |
| Zdroj: | International Journal of Biological Macromolecules. 95:256-262 |
| ISSN: | 0141-8130 |
| DOI: | 10.1016/j.ijbiomac.2016.11.036 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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