Genetic engineering of the Fusarium solani pisi lipase cutinase for enhanced partitioning in PEG-phosphate aqueous two-phase systems

The Fusarium solani pisi lipase cutinase has been genetically engineered to investigate the influence of C-terminal peptide extensions on the partitio ning of the enzyme in PEG-salt based aqueous two-phase bioseparation system s. Seven different cutinase lipase variants were constructed containing var ious C-terminal peptide extensions including tryptophan rich peptide tags ( (WP)(2) and (WP)(4)), positively ((RP)(4)) and negatively ((DP)(4)) charged tags as well as combined tags with tryptophan together with either positiv ely ((WPR)(4)) or negatively ((WPD)(4)) charged amino acids. The modified c utinase variants were stably produced in Escherichia coli as secreted to th e periplasm from which they were efficiently purified by IgG-affinity chrom atography employing an introduced N-terminal IgG-binding ZZ affinity fusion partner present in all variants. Partitioning experiments performed in a P EG 4000/sodium phosphate aqueous two-phase system showed that for variants containing either (WP)(2) or (WP)(4) peptide extensions, 10- to 70-fold inc reases in the partitioning to the PEG rich top-phase were obtained, when co mpared to the wild type enzyme. An increased partitioning was also seen for cutinase variants tagged with both tryptophans and charged amino acids, wh ereas the effect of solely charged peptide extensions was relatively small. In addition, when performing partitioning experiments from cell disintegra tes, the (WP)(4)-tagged cutinase showed a similarly high PEG-phase partitio ning, indicating that the effect from the peptide tag was unaffected by the background of the host proteins. Taken together, the results show that the partitioning of the recombinantly produced cutinase model enzyme could be significantly improved by relatively minor genetic engineering and that the effects observed for purified proteins are retained also in an authentic w hole cell disintegrate system. The results presented should be of general i nterest also for the improvement of the partitioning properties of other in dustrially interesting proteins including bulk enzymes. (C) 2000 Elsevier S cience B.V. All rights reserved.