Bioconversion of D,L‐ tert ‐leucine Nitrile to D‐ tert ‐leucine by Recombinant Cells expressing Nitrile Hydratase and D‐selective Amidase

This project is aimed at studying the feasibility of producing D‐ tert ‐leucine (D‐Tle) from racemic tert ‐leucine‐nitrile (Tle‐nitrile) using nitrile‐transforming enzymes. For the conversion of Tle‐nitrile to D‐Tle, a bacterial strain, previously recovered from a deep‐sea sediment, Rhodococcus erythropolis 870‐AN019, was used, which expresses a nitrile hydratase (NHase)/amidase system for nitrile metabolism. This strain was able to perform the sequential conversion of the nitrile to the acid, via the intermediate racemic tert ‐leucine amide (Tle‐amide). However, the activities of both enzymes were low and the amidase was found to be L‐selective. A developed recombinant E. coli strain, expressing the NHase of R. erythropolis 870‐AN019, showed a 10‐fold increase in activity, compared to the wild type strain. A second recombinant E. coli expressing the D‐selective amidase from Variovorax paradoxus 19–3 was used to complete the conversion of the racemic amide into D‐Tle. The optimal pH and temperature ranges for the enzymes activities were, 7–9 and 30–40 °C, respectively, for the NHase, and 7–9.5 and 47–49 °C, respectively, for the D‐amidase. Thus, a compromise between the optimal biotransformation temperatures was necessary in order to achieve satisfactory activities for both enzymes in one pot reactions. In addition, the D‐amidase activity towards Tle‐amide was 10‐fold lower than the recombinant NHase towards Tle‐nitrile, indicating that a higher concentration of the first enzyme was necessary during biotransformations. The sequential conversion to produce D‐Tle was achieved using a whole cell mixture of both recombinant strains in one pot reactions at different temperatures. A temperature of 35 °C appeared to be the best compromise to obtain good activity of both enzymes in the same reaction. A fed‐batch biotransformation of Tle‐nitrile produced 700 mg of D‐Tle in 4.5 h, however, unconverted nitrile and L‐amide were still present. The accumulation of unconverted amide resulted in the inhibition of the NHase that lead to the accumulation of nitrile, which further inhibited both NHase and D‐amidase. Since both enzymes were inhibited by Tle‐nitrile and Tle‐amide, a continuous process, which would control the concentration of these compounds in the reactor, was investigated. Alginate co‐immobilized whole cells were used in a continuous stirred tank reactor biotransformation to attempt the production of D‐Tle from Tle‐nitrile, but this procedure did not improve the productivity.