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

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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.

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Funding

The work was initiated by the Innovation Center Biriuch, New Technologies, LLC, and was supported within the framework of State Assignment no. 595-00004-18 PR.

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Authors and Affiliations

  1. State Research Institute for Genetics and Selection of Industrial Microorganisms, Kurchatov Institute National Research Center (Kurchatov Institute NRC, GOSNIIGENETIKA), 117545, Moscow, Russia

    S. E. Cheperegin, E. P. Sannikova, A. V. Malysheva, F. A. Klebanov & D. G. Kozlov

Authors
  1. S. E. Cheperegin
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  2. E. P. Sannikova
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  3. A. V. Malysheva
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  4. F. A. Klebanov
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  5. D. G. Kozlov
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Corresponding author

Correspondence to D. G. Kozlov.

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The authors declare that they have no conflicts of interest.

This article does not contain any studies involving animals performed by any of the authors.

This article does not contain any studies involving human participants performed by any of the authors.

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Translated by I. Gordon

Abbreviations: KD—Ca2+ dissociation constant; CL—culture liquid; PAG—polyacrylamide gel; PLA2—phospholipase A2; pla2—gene for PLA2.

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Cheperegin, S.E., Sannikova, E.P., Malysheva, A.V. et al. Highly Active Modified Variants of Recombinant Phospholipase А2 from Streptomyces violaceoruber for Effective Expression in Yeasts. Appl Biochem Microbiol 56, 770–778 (2020). https://doi.org/10.1134/S0003683820070029

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  • DOI: https://doi.org/10.1134/S0003683820070029

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