Molecular Engineering Of L-Aspartate-?-decarboxylase For Catalytic Synthesis Of L-Homophenylalanine

L-Amino acids are very important intermediates for pharmaceuticals,and their synthesis has attracted more and more attention from researchers.The L-aspartate ?-decarboxylase(ABDC)from Pseudomonas dacunhae is a pyridoxal-5'-phosphate(PLP)-dependent enzyme that can catalyze the ?-decarboxylation of L-aspartate to produce L-alanine and carbon dioxide,which has been used in the industrial production of L-alanine.In 1963,Wilson and Komberg first reported that ABDC from Achromobscter could also catalyze the?-decarboxylation of threo-and erythro-?-hydroxy-DL-aspartate to produce L-serine.Inspired by their work,we wondered whether the enzyme could catalyze the other aspartate analogs to produce various L-amino acids.Therefore,this work aims to explore the substrate scope of ABDC,and semi-rational design was adopted to enhance the catalytic efficiency of ABDC for synthesis of L-homophenylalanine and other L-amino acids.Firstly,substrate scope investigation indicates that wild-type ABDC could catalyze the decarboxylation of several 3-substituted aspartates as confirmed by UPLC-MS analysis.Specific activity determination showed that among those substrates,ABDC prefers to catalyze 3(R,S)-hydroxy-DL-aspartic acid with significantly lower specific activity(0.1%)as compared with the natural substrate.Secondly,in order to improve the catalytic efficiency of ABDC towards the unnatural substrates,semi-rational design was applied to ABDC using chemically synthesized and resolved 3(R)-benzyl-L-aspartic acid as a model substrate.The mutant T382G/R37A showing an activity improvement of 15,400-fold in comparison with the wild-type enzyme was obtained.The specific activity of this mutant towards 3(R,S)-methyl-DL-aspartic acid was 3.2-fold that of wild-type enzyme.It is speculated that the main reason is that the small steric residues(G/A)of the mutant provide more space for the accomodation of the substrate bearing bulky substituent at the 3-position.This is consistent with the steady state kinetic constants investigation.Finally,enzymatic preparation of L-homophenylalanine was carried out using the mutant T382G/R37A with an analytic and isolated yield of 73%and 50%,respectively.