Efficient enzymatic synthesis of α-keto acids by redesigned substrate-binding pocket of the l-amino acid deaminase (PmiLAAD)

Abstract In our previous study, we produced α-keto acids by using an L-amino acid deaminase PmiLAAD (wide-type) from Proteus mirabilis, however, the catalytic efficiency was low due to its low substrate affinity. In this study, protein engineering of PmiLAAD was performed to improve the α-keto acid production. PmiLAAD was engineered by iterative CASTing to improve its catalytic performance. The four mutant PmiLAAD-SAVS (PmiLAAD-Phe93Ser-Pro186Ala- Met394Val-Phe184Ser) with 6.6 -fold higher specific activity compared with that of wild-type PmiLAAD has been obtained by high-throughput screening. Comparative kinetics analysis showed that the four mutant PmiLAAD-SAVS had a higher substrate-binding affinity and catalytic efficiency than that of PmiLAAD wild-type. The Km, kcat, and kcat/Km values of the PmiLAAD(SAVS) variant was better (-42.7%, 75.11%, and 85.79%, respectively) than the corresponding values of PmiLAAD wild type. Finally, the whole cell biocatalyst E. coli-pETDuet-1-PmiLAAD(SAVS) has been applied to α-keto acids production. The conversion rate of L-phenylalanine reached 99% by whole-cell biocatalyst E. coli-pETDuet-1-PmiLAAD(SAVS). The conversion of (D/L)-4-phenylalanine was reached 49.5% after 7 h by whole-cell biocatalyst E. coli-pETDuet-1-PmiLAAD(SAVS), while the conversion of E. coli-pETDuet-1-PmiLAAD (wild type) was only 18% after an extension of the reaction time (24 h). This study has developed a robust whole-cell E. coli biocatalyst for α-keto acids production by protein engineering, and this strategy may be useful for the construction of other biotransformation biocatalysts.