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 (PmiLAAO) from Proteus mirabilis was discovered by data mining. Secondly, by gene expression vector screening, pETDuet-1-PmiLAAO activity improved by 130%, as compared to the pET20b-PmiLAAO. PmiLAAO production was increased to 9.8 U ml-1 by optimized expression condition (OD600 = 0.65, 0.45 mmol l-1 IPTG, 20 h of induction). Furthermore, The PmiLAAO 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 PmiLAAO were 6.5 and 37°C, respectively. Afterwards, in order to simplify product separation process, E. coli-pETduet-1-PmiLAAO 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-PmiLAAO. 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.
Keywords: amino acids; biocatalysis; biotransformation; l-amino acid oxidases; α-keto acid.