High yield production and purification of two recombinant thermostable phosphotriesterase-like lactonases from Sulfolobus acidocaldarius and Sulfolobus solfataricus useful as bioremediation tools and bioscavengers

Autor: Chiara Schiraldi, Luigi Fedele, Ilaria Scognamiglio, Mario De Rosa, Elena Porzio, Giuseppe Manco, Odile Francesca Restaino, Alberto Alfano, Maria Giovanna Borzacchiello
Přispěvatelé: Restaino, Odile Francesca, Borzacchiello, Maria Giovanna, Scognamiglio, Ilaria, Fedele, Luigi, Alfano, Alberto, Porzio, Elena, Manco, Giuseppe, De Rosa, Mario, Schiraldi, Chiara
Rok vydání: 2018
Předmět:
0106 biological sciences
0301 basic medicine
Sulfolobus acidocaldarius
lcsh:Biotechnology
Archaeal Proteins
ved/biology.organism_classification_rank.species
Ultra-filtration membrane-based purification
Ultrafiltration
Biology
Protein Engineering
01 natural sciences
Extremozymes
Fed-batch fermentation
03 medical and health sciences
chemistry.chemical_compound
Bioremediation
Organophosphate
lcsh:TP248.13-248.65
010608 biotechnology
Enzyme Stability
Escherichia coli
Chemical Precipitation
Response surface methodology
chemistry.chemical_classification
ved/biology
Sulfolobus solfataricus
Archaea
Organophosphates
Recombinant Proteins
Biodegradation
Environmental

Phosphoric Triester Hydrolases
030104 developmental biology
Enzyme
chemistry
Biochemistry
Batch Cell Culture Techniques
Galactose
Yield (chemistry)
Fermentation
Thermostable phosphotriesterase-like lactonase
Chromatography
Gel

Extremozyme
Research Article
Biotechnology
Zdroj: BMC Biotechnology
BMC Biotechnology, Vol 18, Iss 1, Pp 1-15 (2018)
ISSN: 1472-6750
DOI: 10.1186/s12896-018-0427-0
Popis: Background Thermostable phosphotriesterase-like lactonases (PLLs) are able to degrade organophosphates and could be potentially employed as bioremediation tools and bioscavengers. But nowadays their manufacturing in high yields is still an issue that limits their industrial applications. In this work we aimed to set up a high yield production and purification biotechnological process of two recombinant PLLs expressed in E. coli, the wild type SacPox from Sulfolobus acidocaldarius and a triple mutated SsoPox C258L/I261F/W263A, originally from Sulfolobus solfataricus. To follow this aim new induction approaches were investigated to boost the enzyme production, high cell density fermentation strategies were set-up to reach higher and higher enzyme yields up to 22-L scale, a downstream train was studied to meet the requirements of an efficient industrial purification process. Results Physiological studies in shake flasks demonstrated that the use of galactose as inducer increased the enzyme concentrations up to 4.5 folds, compared to the production obtained by induction with IPTG. Optimising high cell density fed-batch strategies the production and the productivity of both enzymes were further enhanced of 26 folds, up to 2300 U·L− 1 and 47.1 U·L− 1·h− 1 for SacPox and to 8700 U·L− 1 and 180.6 U·L− 1·h− 1 for SsoPox C258L/I261F/W263A, and the fermentation processes resulted scalable from 2.5 to 22.0 L. After being produced and extracted from the cells, the enzymes were first purified by a thermo-precipitation step, whose conditions were optimised by response surface methodology. A following ultra-filtration process on 100 and 5 KDa cut-off membranes drove to a final pureness and a total recovery of both enzymes of 70.0 ± 2.0%, suitable for industrial applications. Conclusions In this paper, for the first time, a high yield biotechnological manufacturing process of the recombinant enzymes SacPox and SsoPox C258L/I261F/W263A was set-up. The enzyme production was boosted by combining a new galactose induction approach with high cell density fed-batch fermentation strategies. An efficient enzyme purification protocol was designed coupling a thermo-precipitation step with a following membrane-based ultra-filtration process. Electronic supplementary material The online version of this article (10.1186/s12896-018-0427-0) contains supplementary material, which is available to authorized users.
Databáze: OpenAIRE
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