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Optimization of the Expression of Nitrilase from Alcaligenes denitrificans in Rhodococcus rhodochrous to Improve the Efficiency of Biocatalytic Synthesis of Ammonium Acrylate

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Abstract

A Rhodococcus rhodochrous strain, M33-2nit, has been constructed with two copies of the nitrilase gene from A. denitrificans В-9582 under the control of nitrile hydratase promoter from R. rhodochrous M8. The optimized cultivation of this strain made it possible to obtain the enzyme in a concentration of up to 17 g of dry cells/L with a specific activity of up to 7 U/mg cdw with a two-substrate culturing scheme in a fed-batch reactor with the sequential addition of glucose and acetate. The capacities of A. denitriificans В-9582 and R. rhodochrous M33-2nit cells to synthesize ammonium acrylate from acrylonitrile under conditions imitating industrial synthesis are compared. It is shown that R rhodochrous M33-2nit cells can synthesize ammonium acrylate under higher rates of acrylonitrile feeding than A. denitriificans В-9582 cells. The potential to obtain a highly concentrated solution of ammonium acrylate (450 g/L) with R. rhodochrous M33-2nit cells as a biocatalyst was demonstrated. The conversion of acrylonitrile to ammonium acrylate reached 99.5%.

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Funding

This work was supported by the Russian Science Foundation (project no. 16-14-00216, “Study on cobalt-dependent gene expression in Rhodococcus bacteria and the construction on its basis of a platform for the biosynthesis of enzyme biocatalysts to obtain acrylic monomers”).

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Authors and Affiliations

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

    K. V. Lavrov, E. G. Grechishnikova, A. O. Shemyakina, A. D. Novikov, T. I. Kalinina & A. S. Yanenko

  2. Scientific Production Center Armbiotechnology, National Academy of Sciences of Armenia, 0056, Yerevan, Armenia

    A. S. Epremyan

  3. BIOAMIDE Closed Joint Stock Venture, 410033, Saratov, Russia

    S. A. Glinskii, R. A. Minasyan & S. P. Voronin

Authors
  1. K. V. Lavrov
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  2. E. G. Grechishnikova
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  3. A. O. Shemyakina
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  4. A. D. Novikov
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  5. T. I. Kalinina
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  6. A. S. Epremyan
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  7. S. A. Glinskii
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  8. R. A. Minasyan
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  9. S. P. Voronin
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  10. A. S. Yanenko
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Correspondence to K. V. Lavrov or A. S. Yanenko.

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The authors declare that they have no conflict of interest. This article does not contain any studies involving animals or human participants performed by any of the authors.

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The electronic version of the article contains Supplementary Materials, which are available free of charge at the website of the journal Biotechnology, http://www.biotechnology-journal.ru.

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Translated by I.Gordon

Abbreviations: cdw—cell dry weight; EDTA, ethylene diamine tetraacetic acid; GS, gas chromatography; HPLC, high-performance liquid chromatography; LB medium, lysogeny broth medium; MS medium, mineral synthetic medium; OD600, optical density at wavelength of 600 nm; PAGE, polyacrylamide gel electrophoresis; SD, standard deviation; SDS, sodium dodecyl sulfate.

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Lavrov, K.V., Grechishnikova, E.G., Shemyakina, A.O. et al. Optimization of the Expression of Nitrilase from Alcaligenes denitrificans in Rhodococcus rhodochrous to Improve the Efficiency of Biocatalytic Synthesis of Ammonium Acrylate. Appl Biochem Microbiol 55, 861–869 (2019). https://doi.org/10.1134/S0003683819090035

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