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α-keto acids are compounds of primary interest for the fine chemical, pharmaceutical and agrochemical sectors.  l -amino acid oxidases as an efficient tool are used for α-keto acids preparation in this study. Firstly, an  l -amino acid oxidase ( Pmi LAAO) from  Proteus mirabilis  was discovered by data mining .  Secondly, by gene expression vector screening, pETDuet-1- Pmi LAAO activity improved by 130%, as compared to the pET20b- Pmi LAAO.  Pmi LAAO production was increased to 9.8 U ml −1  by optimized expression condition (OD 600  = 0.65, 0.45 mmol l −1  IPTG, 20 h of induction). Furthermore, The  Pmi LAAO was stabile in the pH range of 4.0–9.0 and in the temperature range of 10–40°C; the optimal pH and temperature of recombinant  Pmi LAAO were 6.5 and 37°C, respectively. Afterwards, in order to simplify product separation process,  E. coli -pETduet-1- Pmi LAAO was immobilized in Ca-alginate beads. Continuous production of 2-oxo-3-phenylpropanoic acid was conducted in a packed-bed reactor via immobilized  E. coli -pETduet-1- Pmi LAAO. Significantly, 29.66 g l −1  2-oxo-3-phenylpropanoic acid with a substrate conversion rate of 99.5% was achieved by correspondingly increasing the residence time (25 h). This method holds the potential to be used for efficiently producing pure α-keto acids.

Efficient production of α-keto acids by immobilized E. coli -pETduet-1- Pmi LAAO in a jacketed packed-bed reactor