초록
<P><B>Background</B></P><P>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 <I>E. coli</I>, the <I>wild type Sac</I>Pox from <I>Sulfolobus acidocaldarius</I> and a triple mutated <I>Sso</I>Pox C258L/I261F/W263A, originally from <I>Sulfolobus solfataricus.</I> 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.</P><P><B>Results</B></P><P>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<SUP>− 1</SUP> and 47.1 U·L<SUP>− 1</SUP>·h<SUP>− 1</SUP> for <I>Sac</I>Pox and to 8700 U·L<SUP>− 1</SUP> and 180.6 U·L<SUP>− 1</SUP>·h<SUP>− 1</SUP> for <I>Sso</I>Pox 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.</P><P><B>Conclusions</B></P><P>In this paper, for the first time, a high yield biotechnological manufacturing process of the recombinant enzymes <I>Sac</I>Pox and <I>Sso</I>Pox 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.</P><P><B>Electronic supplementary material</B></P><P>The online version of this article (10.1186/s12896-018-0427-0) contains supplementary material, which is available to authorized users.</P>