Continuous colorimetric assay that enables high-throughput screening of N-acetylamino acid racemases.
Continuous colorimetric assay that enables high-throughput screening of N-acetylamino acid racemases.
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DOI：
10.1021/ac5047328
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发表时间：
2015-03
影响因子：
7.4
通讯作者：
Guiomar Sánchez-Carrón;T. Fleming;Karen E. Holt-Tiffin;D. Campopiano
中科院分区：
文献类型：
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作者：
Guiomar Sánchez-Carrón;T. Fleming;Karen E. Holt-Tiffin;D. Campopiano
n -乙酰基氨基酸消旋酶（NAAARs）在酶促合成具有重要生物技术意义的手性氨基酸分子方面已显示出其潜力。为了确定新的活性并通过工程方法改进这些酶，需要合适的筛选方法。以前从Amycolatopsis Ts-1-60中提取NAAAR的工程是依靠一种体内选择系统实现的，该系统将大肠杆菌l -甲硫氨酸营养不良菌的生存能力与改进后的酶的活性联系起来。然而，该试验仅适用于筛选n-乙酰- d -蛋氨酸，因此限制了该酶向其他天然或非天然乙酰化氨基酸进化的潜力。在这里，我们报告了一种基于分光光度微滴板的NAAAR测定方法的优化和应用。该分析是基于检测由l -氨基酸酰化酶水解n -酰化底物和随后由fad依赖的l -氨基酸氧化酶（L-AAO）氧化形成的氨基酸反应产物。L-AAO的辅因子循环导致过氧化氢的形成，这很容易使用辣根过氧化物酶（HRP）和邻二苯胺进行监测。该方法测定了NAAAR的动力学参数，并鉴定出n -乙酰- d -萘丙氨酸是一种新的NAAAR底物。这种稳健的方法也适用于直接从细胞裂解物中高通量筛选NAAAR突变基因文库。
N-Acetyl amino acid racemases (NAAARs) have demonstrated their potential in the enzymatic synthesis of chiral amino acids, molecules of significant biotechnology interest. In order to identify novel activities and to improve these enzymes by engineering approaches, suitable screening methods are necessary. Previous engineering of the NAAAR from Amycolatopsis Ts-1-60 was achieved by relying on an in vivo selection system that linked the viability of an E. coli L-methionine auxotroph to the activity of the improved enzyme. However, this assay was only suitable for the screening of N-acetyl-D-methionine, therefore limiting the potential to evolve this enzyme toward other natural or non-natural acetylated amino acids. Here, we report the optimization and application of a spectrophotometric microtiter-plate-based assay for NAAAR. The assay is based on the detection of the amino acid reaction product formed by hydrolysis of the N-acylated substrate by an L-amino acid acylase and its subsequent oxidation by an FAD-dependent L-amino acid oxidase (L-AAO). Cofactor recycling of the L-AAO leads to the formation of hydrogen peroxide which is easily monitored using horseradish peroxidase (HRP) and o-dianisidine. This method allowed for the determination of the kinetic parameters of NAAAR and led to the identification of N-acetyl-D-naphthylalanine as a novel NAAAR substrate. This robust method is also suitable for the high-throughput screening of NAAAR mutant gene libraries directly from cell lysates.