Expanding the Enzymatic Toolbox for β-amino Acids and Unnatural Amino Acids Manufacturing

β-Amino acids, characterized by the separation of their terminal carboxylic acid and
amino groups by two carbon atoms (Cα and Cβ), exhibit remarkable structural
versatility. This arrangement allows for R or S isomers at both Cα and Cβ positions,
resulting in up to four diastereomers for a given side chain. Such diversity facilitates
the generation of a broad array of stereo- and regio-isomers, alongside the potential
for di-substitution, making β-amino acids highly valuable in molecular design. Their
incorporation into peptidomimetics has led to compounds with potent biological
activity and enhanced resistance to proteolysis. Furthermore, β-amino acids and their
derivatives serve as essential chiral building blocks in pharmaceutical synthesis,
highlighting their significance in drug discovery and development.
This study focuses on enzymatic approaches to β-amino acid production as a
sustainable alternative to traditional chemical synthesis, which often suffers from low
carbon efficiency. Leveraging both wildtype and engineered variants of aspartase and
3-methylaspartate ammonia-lyase (MAL), this project explores their catalytic
potential for industrial applications. Aspartase catalyzes the reversible deamination of
L-aspartic acid via a carbanion mechanism to produce fumaric acid and ammonium
ion, while MAL facilitates the α,β-elimination of ammonia from 3-methylaspartate to
form mesaconate.
Novel aspartase and MAL enzymes sourced from thermophilic organisms were
identified and characterized. Protocols for their recombinant expression and
purification were established, and their enzymatic activities were validated through
NMR and spectrometric assays. This research provides critical insights into the
catalytic mechanisms and industrial applicability of aspartase and MAL, offering a
foundation for environmentally friendly and efficient production of β-amino acids and
other unnatural amino acids.