Bioconversion of D,L-tert-leucine nitrile to D-tert-leucine by recombinant cells expressing nitrile hydratase and D-selective amidase

Abstract

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 While 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. © 2004 Wiley-VCH Verlag GmbH & Co. KGaA.