Highly Active Modified Variants of Recombinant Phospholipase А2 from Streptomyces violaceoruber for Effective Expression in Yeasts

Abstract

The ability of yeast to produce highly active variants of the recombinant phospholipase PLA2 is confirmed. Highly active variants were based on an original enzyme from the Streptomyces violaceoruber А-2688 strain. Various approaches to reduce enzyme toxicity and increase its expression, including point mutations, the construction of artificial N- and/or C-end proregions, and the inactivation of glycosylation sites, were tested. The main result was the obtainment of modified PLA2 enzymes with the same secretion level as their weakly active prototype but a specific activity that was at least ten times higher. As the main feature, the selected mutants were characterized by a lower affinity for Ca2+, which probably accounts for their low toxicity at the stage of biosynthesis and the possibility of their activation under special conditions, e.g. by the addition of calcium during incubation with egg yolk. It appears that the obtained variants can significantly reduce the cost of the use of PLA2 enzyme preparations in industries that allow the use of high calcium concentrations.