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[摘要]：The gene encoding aspartate ammonia lyase (aspB) from Bacillus sp. YM55-1 has been cloned and over-expressed, and the recombinant enzyme contg. a C-terminal His6 tag has been purified to homogeneity and subjected to kinetic characterization. Kinetic studies have shown that the His6 tag does not affect AspB activity. The enzyme processes L-aspartic acid, but not D-aspartic acid, with a Km of u15 mM and a kcat, of u40 s-1. By using this recombinant enzyme in the reverse reaction, a set of four N-substituted aspartic acids were prepd. by the Michael addn. of hydroxylamine, hydrazine, methoxylamine, and methylamine to fumarate. Both hydroxylamine and hydrazine were found to be excellent substrates for AspB. The kcat,, values are comparable to those obsd. for the AspB-catalyzed addn. of ammonia to fumarate (u90 s-1), whereas the Km values are only slightly higher. The products of the enzyme-catalyzed addn. of hydrazine, methoxylamine, and methylamine to fumarate were isolated and characterized by NMR spectroscopy and HPLC anal., which revealed that AspB catalyzes all the addns. 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. |
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