Efficient reductive amination process for enantioselective synthesis of L-phosphinothricin applying engineered glutamate dehydrogenase

Abstract

The objective of this study was to identify and exploit a robust biocatalyst that can be applied in reductive amination for enantioselective synthesis of the competitive herbicide L-phosphinothricin. Applying a genome mining-based library construction strategy, eight NADPH-specific glutamate dehydrogenases (GluDHs) were identified for reductively aminating 2-oxo-4-[(hydroxy)(methyl)phosphinoyl]butyric acid (PPO) to L-phosphinothricin. Among them, the glutamate dehydrogenase cloned from Pseudomonas putida (PpGluDH) exhibited relatively high catalytic activity and favorable soluble expression. This enzyme was purified to homogeneity for further characterization. The specific activity of PpGluDH was 296.1 U/g-protein, which is significantly higher than the reported value for a GluDH. To the best of our knowledge, there has not been any report on protein engineering of GluDH for PPO-oriented activity. Taking full advantage of the available information and the diverse characteristics of the enzymes in the enzyme library, PpGluDH was engineered by site-directed mutation based on multiple sequence alignment. The mutant I170M, which had 2.1-fold enhanced activity, was successfully produced. When the I170M mutant was applied in the batch production of L-phosphinothricin, it showed markedly improved catalytic efficiency compared with the wild type enzyme. The conversion reached 99% (0.1 M PPO) with an L-phosphinothricin productivity of 1.35 g/h·L, which far surpassed the previously reported level. These results show that PpGluDH I170M is a promising biocatalyst for highly enantioselective synthesis of L-phosphinothricin by reductive amination.

Rational Molecular Engineering of Glutamate Dehydrogenases for Enhancing Asymmetric Reductive Amination of Bulky α‐Keto Acids

“…The bulky α‐keto acid PPO is a precursor in the synthesis of L‐PPT by asymmetric reductive amination. In our previous work, we identified a NADP + ‐specific GluDH ( Pp GluDH) from Pseudomonas putida that was able to catalyze the reductive amination of PPO to L‐PPT. This GluDH was engineered by site‐directed mutagenesis based on multiple sequence alignment.…”

“…The recombinant GluDHs used in this work were constructed in our previous work and were maintained in our laboratory. PPO, 2‐oxoheptanoic acid (S8), 2‐oxooctanoic acid (S9) and 2‐oxononanoic acid (S10) were synthesized in our laboratory and were characterized by MS and NMR (Figures S1–S8).…”

“…The reaction was conducted at 35 °C for 10 min, with shaking at 600 rpm, and then quenched by the addition of 40 μL 5 M NaOH. The amount of product formed was then determined by HPLC, as described previously . One unit of enzyme activity in the reductive amination was defined as the amount of enzyme catalyzing the formation of 1 μmol L‐PPT per min at 35 °C.…”

“…The kinetic parameters were determined from three independent initial rate measurements performed with the same batch of purified enzyme over 0.04–0.4 M PPO with 0.5 M NH 4 + ((NH 4 ) 2 SO 4 ) and 10 mM NADPH. The initial rate was measured by HPLC analysis, as described previously …”

“…The amount of product formed was then determined by HPLC, as described previously. [11] One unit of enzyme activity in the reductive amination was defined as the amount of enzyme catalyzing the formation of 1 mmol L-PPT per min at 35 8C.…”