Characterization of Phenylalanine Ammonia Lyases from Lettuce (Lactuca sativa L.) as Robust Biocatalysts for the Production of d- and l-Amino Acids

Abstract

Phenylalanine ammonia lyase (PAL) catalyzes the reversible conversion of L-phenylalanine into the corresponding trans-cinnamic acid, providing a route to optically pure α-amino acids. We explored the catalytic function of all five PALs encoded in the genome of lettuce (Lactuca sativa L.) that are previously known to be involved in wound browning. All LsPALs were active toward L-phenylalanine in the ammonia elimination reaction and displayed maximum activity at 55−60 °C and pH 9.0−9.5. However, four of them, LsPAL1−LsPAL4, showed significantly higher activity and thermal stability than LsPAL5, as well as a broader substrate spectrum including some challenging substrates with steric demanding or electron-donating substituents. The best one LsPAL3 was subjected to the kinetic resolution of a panel of 21 rac-phenylalanine derivatives, as well as the ammonia addition of 21 cinnamic acid derivatives. It showed excellent enantioselectivity in most cases and significantly better activity than previously described PALs for a number of challenging non-natural substrates, demonstrating its great potential in biocatalysis.

The Biocatalytic Potential of Aromatic Ammonia–Lyase from Loktanella atrilutea

“…Residue N199 is highly specific to the novel AAL subclass represented by La AAL and AL‐11, in other PALs, PAL/TALs hydrophobic residues, Leu, Val or Met are found at the homologue sequence position (Figure 7B). In accordance, Pc PAL, At PAL, Rt PAL, Pza PAL and Ls PAL3 possessing Val at this position (Figure 7B), transformed with good conversion the m –methyl–cinnamic acid [2a,c,10c,18] . Thus, we suppose that linear and polar substituents, as in case of substrates 2 n , 2 q , can be embedded and oriented towards N199 and N200, while the sterically more demanding (in case of 2 o and 2 p ) or non–polar substituents (in case of 2 l ) provide less favourable orientations in this catalytic environment (Figure S35).…”