Enzymatic Synthesis of a Key Intermediate for Rosuvastatin by Nitrilase-Catalyzed Hydrolysis of Ethyl (R)-4-Cyano-3-hydroxybutyate at High Substrate Concentration

Abstract

An enzymatic method for the synthesis of ethyl (R)-3-hydroxyglutarate from ethyl (R)-4-cyano-3-hydroxybutyate was developed by using free and immobilized recombinant Escherichia coli BL21(DE3)pLysS harboring a nitrilase gene from Arabidopsis thaliana (AtNIT2). The hydrolysis of ethyl (R)-4-cyano-3-hydroxybutyate proceeded with the freely suspended cells of the biocatalyst under the optimized conditions of 1.5 mol L−1 (235.5 g L−1) substrate concentration and 6.0 wt % loading of wet cells at pH 8.0 and 25 °C, with 100 % conversion obtained in 4.5 h. Furthermore, immobilization of the whole cells enhanced their substrate tolerance, stability, and reusability. Under the optimized conditions (100 mmol L−1 tris(hydroxymethyl)aminomethane hydrochloride buffer, pH 8.0, 25 °C), the immobilized biocatalyst could be reused for up to 16 batches, with a biocatalyst productivity of 55.6 g gwet cells−1 and a space-time productivity of 625.5 g L−1 d−1. These results demonstrated that the immobilized whole cells might be used as a biocatalyst in the industrial production of ethyl (R)-3-hydroxyglutarate, a key intermediate for the synthesis of rosuvastatin.