Abstract
BackgroundThermostable 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.ResultsPhysiological 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.ConclusionsIn 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.
Highlights
Thermostable phosphotriesterase-like lactonases (PLLs) are able to degrade organophosphates and could be potentially employed as bioremediation tools and bioscavengers
The design of large-scale, high yield biotechnological production and purification processes for most of these PLL enzymes is still an unresolved issue. In this experimental work we focused our attention on two enzymes, the wild type SacPox from S. acidocaldarius and a triple mutated form of SsoPox (SsoPox 3 M, with the mutated residues C258L/I261F/W263A) both previously isolated, in case of SsoPox 3 M modified by applying in vitro protein evolution strategies, and expressed in soluble forms in Escherichia coli BL21
First physiological experiments were run in shake flasks to study the kinetic of growth of the two recombinant strains and to investigate the best induction conditions by using different concentrations of IPTG or of galactose, used as replacement of IPTG (Fig. 2a-c)
Summary
Thermostable phosphotriesterase-like lactonases (PLLs) are able to degrade organophosphates and could be potentially employed as bioremediation tools and bioscavengers. PLL enzymes could be potentially employed as biocatalysists in OP degradation and removal in different fields: in environmental bioremediation as friendly and economic tools in alternative to chemical or physical decontamination approaches, in food safety and industry to remove pesticides from groceries and fruits, in public health and defense as valid exogenous bioscavengers in small defense systems and as innovative biomedical countermeasures for the quick sequester and inactivation of OPs in case of human exposure [1,2,3, 13,14,15,16,17,18,19] Thanks to their extremely high thermal stability, their resistance to organic solvents, their ability to operate in wide pH ranges, in different buffers and in harsh conditions like in presence of surfactants and in outdoor, as well for the possibility of long-term storage at room temperatures, these extremozymes demonstrated to be more industrially attractive than their mesophilic counterparts, such as the phosphotriesterase enzymes (PTEs) from Flavobacterium sp. Thanks to their extremely high thermal stability, their resistance to organic solvents, their ability to operate in wide pH ranges, in different buffers and in harsh conditions like in presence of surfactants and in outdoor, as well for the possibility of long-term storage at room temperatures, these extremozymes demonstrated to be more industrially attractive than their mesophilic counterparts, such as the phosphotriesterase enzymes (PTEs) from Flavobacterium sp. strains, Brevundimonas diminuta, Pseudomonas a b
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