Enhancing a Sphaerobacter thermophilus ω-transaminase for kinetic resolution of β- and γ-amino acids

Sphaerobacter thermophilus synthesizes an omega-transaminase (omega-TA) that allows the production of enantiomerically pure beta-amino acids. To obtain omega-TA variants with a higher activity and more favorable properties for industrial use, we modified critical amino acid residues either in the catalytic center or in a previously proposed signature motif critical for aromatic beta-amino acid omega-TAs. Seventeen different variants of this enzyme were generated and their activity was examined with four beta-amino acids and one gamma-amino acid, and compared with the wildtype's activity. Among all variants, seven showed up to ninefold higher activity with at least one of the tested substrates. For most of these seven variants, the temperature optimum was even lower as in the wild type enzyme, with keeping a high temperature stability, making them more valuable for industrial purposes. Our results indicate that for the production of enantiomerically pure beta-amino acids replacement of critical amino acid residues in the proposed signature motif of omega-TAs is a more effective strategy than modifying their catalytic center. Another finding was, that the proposed motif is not only suitable for aromatic amino acid omega-TAs, because some of the variants have a higher activity with beta-alanine or beta-leucine than with aromatic beta-amino acids.