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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