Discovery of amino acid amide racemase and its use in the dynamic kinetic resolution of amino acid amides

1.Achromobacter obae的α-氨基己内酰胺(ACL)外消旋酶具有氨基酸酰胺外消旋活性。从不同来源的微生物中分离出D-氨基肽酶(DAP)、D-氨基酸酰胺酶(DAA)等作用于不同氨基中间体的氨基酸酰胺酶，并对其一级结构进行了推导，并在大肠杆菌中进行了表达。以人Ochrobactrumc1-38的D-氨基肽酶和乙酰胆碱外消旋酶为底物，研究了以L丙氨酸酰胺为原料的酶促合成D-丙氨酸的反应。后续的酶促反应结果表明，ACL外消旋酶与DAP的结合可用于DL-氨基酸酰胺的动态动力学拆分，合成D-氨基酸。DAA的结构和ACL消旋酶的定向进化：我们建立了一种用立体特异性氨基酸酰胺酶、氨基酸氧化酶和4-氨基安替比林测定ACL消旋酶活性的比色法。人嗜铬杆菌SV3的D-氨基酸酰胺酶的结构在2.1a分辨率下被解析。

1. Amino acid amide racemizing activity was discovered in alpha-amino-epsilon-caprolactam (ACL) racemase from Achromobacter obae.2. Amino acid amidases acting for various amino mid amides, such as D-aminopeptidase (DAP), D-amino acid amidase (DAA), were isolated from various microbial sources, their primary structures deduced and expression in E. coli were made possible.3. The enzymatic synthesis of D-alanin from L-alanine amide has been demonstrated by use of D-aminopeptidase (DAP) from Ochrobactrum anthropi C1-38 and ACL racemase. The result of successive enzymatic reaction shows that the combination of ACL racemase and DAP can be applied for dynamic kinetic resolution of DL-amino acid amides to yield D-amino acids.4. Structure of DAA and directed evolution of ACL racemase: We developed a colorimetric method to measure the activity of ACL racemase with stereospecific amino acid amidase and amino acid oxidase, and 4-aminoantipyrine. The structure of D-amino acid amidase from Ochrobactrum anthropiSV3 was solved at 2.1A resolution.