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

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