Biocatalytic Enantioselective Synthesis of N‐Substituted Aspartic Acids by Aspartate Ammonia Lyase

Abstract The gene encoding aspartate ammonia lyase ( aspB ) from Bacillus sp. YM55‐1 has been cloned and overexpressed, and the recombinant enzyme containing a C‐terminal His 6 tag has been purified to homogeneity and subjected to kinetic characterization. Kinetic studies have shown that the His 6 tag does not affect AspB activity. The enzyme processes L ‐aspartic acid, but not D ‐aspartic acid, with a K m of ≈15 m M and a k cat of ≈40 s −1 . By using this recombinant enzyme in the reverse reaction, a set of four N ‐substituted aspartic acids were prepared by the Michael addition of hydroxylamine, hydrazine, methoxylamine, and methylamine to fumarate. Both hydroxylamine and hydrazine were found to be excellent substrates for AspB. The k cat values are comparable to those observed for the AspB‐catalyzed addition of ammonia to fumarate (≈90 s −1 ), whereas the K m values are only slightly higher. The products of the enzyme‐catalyzed addition of hydrazine, methoxylamine, and methylamine to fumarate were isolated and characterized by NMR spectroscopy and HPLC analysis, which revealed that AspB catalyzes all the additions with excellent enantioselectivity (>97 % ee ). Its broad nucleophile specificity and high catalytic activity make AspB an attractive enzyme for the enantioselective synthesis of N ‐substituted aspartic acids, which are interesting building blocks for peptide and pharmaceutical synthesis as well as for peptidomimetics.