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

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%.