Glycine oxidase based high-throughput solid-phase assay for substrate profiling and directed evolution of (R)- and (S)-selective amine transaminases.
Glycine oxidase based high-throughput solid-phase assay for substrate profiling and directed evolution of (R)- and (S)-selective amine transaminases.
复制标题
基于甘氨酸氧化酶的高通量固相测定，用于 (R)-和 (S)-选择性胺转氨酶的底物分析和定向进化。
作者：
M. Weiß;Ioannis V. Pavlidis;Clare Vickers;M. Höhne;U. Bornscheuer
转氨酶是手性胺合成生物催化工具箱中最重要的酶之一，因为它们可以实现定量产率和高对映选择性的不对称合成。为了能够对转氨酶进行底物分析以接受不同的胺，开发了甘氨酸氧化酶和辣根过氧化物酶偶联测定。当胺基从氨基供体底物转移到乙醛酸时检测转氨酶活性，产生甘氨酸，随后被甘氨酸氧化酶氧化，进而释放过氧化氢。辣根过氧化物酶利用过氧化氢产生苯醌，苯醌通过随后的缩合反应形成红色醌亚胺染料。由于甘氨酸不携带手性中心，接受乙醛酸作为氨基受体的 (R)-和 (S)-选择性转氨酶都适合筛选。该原理已被转移以建立高通量固相测定，该测定极大地减少了转氨酶定向进化的筛选工作，因为仅选择活性变体进行进一步分析。
Transaminases represent one of the most important enzymes of the biocatalytic toolbox for chiral amine synthesis as they allow asymmetric synthesis with quantitative yields and high enantioselectivity. In order to enable substrate profiling of transaminases for acceptance of different amines, a glycine oxidase and horseradish peroxidase coupled assay was developed. Transaminase activity is detected upon transfer of an amine group from an amino donor substrate to glyoxylate, generating glycine, which is subsequently oxidized by glycine oxidase, releasing hydrogen peroxide in turn. Horseradish peroxidase uses the hydrogen peroxide to produce benzoquinone, which forms a red quinone imine dye by a subsequent condensation reaction. As glycine does not carry a chiral center, both (R)- and (S)-selective transaminases accepting glyoxylate as amino acceptor are amenable to screening. The principle has been transferred to establish a high-throughput solid-phase assay which dramatically decreases the screening effort in directed evolution of transaminases, as only active variants are selected for further analysis.