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

摘要

Tie gene encoding aspartate ammonia lyase (aspB) from Bacillus sp. YM55-1 has been cloned and over-expressed, and the recombinant enzyme containing a C-terminal His, 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,, of approximate to 15 mm and a k(cat) of approximate to 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 (approximate to 90s(-1)), whereas the K-cat values are only slightly higher. The products of the enzymecatalyzed 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.