Bioconversion of L-glutamic acid to alpha-ketoglutaric acid by an immobilized whole-cell biocatalyst expressing L-amino acid deaminase from Proteus mirabilis

摘要

The goal of this work was to develop an immobilized whole-cell biocatalytic process for the environment-friendly synthesis of alpha-ketoglutaric acid (alpha-KG) from L-glutamic acid. We compared the suitability of Escherichia coli and Bacillus subtilis strains overexpressing Proteus mirabilis L-amino acid deaminase (L-AAD) as potential biocatalysts. Although both recombinant strains were biocatalytically active, the performance of B. subtilis was superior to that of E. coli. With L-glutamic acid as the substrate, alpha-KG production levels by membranes isolated from B. subtilis and E. coli were 55.3 +/- 1.73 and 21.7 +/- 0.39 mu g/mg protein/min, respectively. The maximal conversion ratio of L-glutamic acid to alpha-KG was 31% (w/w) under the following optimal conditions: 15 g/L L-glutamic acid, 20 g/L whole-cell biocatalyst, 5 mM MgCl2, 40 degrees C, pH 8.0, and 24-h incubation. Immobilization of whole cells with alginate increased the recyclability by an average of 23.33% per cycle. This work established an efficient one-step biotransformation process for the production of alpha-KG using immobilized whole B. subtilis overexpressing P. mirabilis L-AAD. Compared with traditional multistep chemical synthesis, the biocatalytic process described here has the advantage of reducing environmental pollution and thus has great potential for the large-scale production of alpha-KG