Continuous colorimetric screening assay for detection of D-amino acid aminotransferase mutants displaying altered substrate specificity
Continuous colorimetric screening assay for detection of D-amino acid aminotransferase mutants displaying altered substrate specificity
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DOI：
10.1016/j.ab.2014.06.006
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发表时间：
2014-10-15
影响因子：
2.9
通讯作者：
Chica, Roberto A.
中科院分区：
文献类型：
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作者：
Barber, Janet E. B.;Damry, Adam M.;Chica, Roberto A.
D-氨基酸氨基转移酶（DAAT）催化许多D-氨基酸的合成，使其成为生产对映体纯D-氨基酸的有吸引力的生物催化剂。为了支持其生物催化适用性，需要对非天然底物显示增加的活性的改进的变体。在这里，我们报告了一个高通量，比色，连续偶联酶分析的DAAT突变体库的筛选，是基于使用D-氨基酸氧化酶（DAAO）的发展。在本试验中，DAAT的D-氨基酸产物被DAAO氧化，同时释放过氧化氢，通过加入辣根过氧化物酶和D-联茴香胺进行比色检测。使用该测定法，我们测量了DAAT的表观K-M和k（cat）值，并通过在96孔板中筛选细胞裂解物鉴定了显示底物特异性改变的突变体。DAAO偶联测定是灵敏的，因为其允许检测相对于野生型显示k（cat）/K-M降低约2000倍的DAAT突变体。此外，DAAO测定能够鉴定两种DAAT突变体（V33 Y和V33 G），其在转氨基作用非天然受体苯丙酮酸方面比野生型更有效。我们预期，该测定将可用于对非天然底物显示增加的活性的其他突变体的工程化。(C)2014 Elsevier Inc. All rights reserved.
D-Amino acid aminotransferase (DAAT) catalyzes the synthesis of numerous D-amino acids, making it an attractive biocatalyst for the production of enantiopure D-amino acids. To bolster its biocatalytic applicability, improved variants displaying increased activity toward non-native substrates are desired. Here, we report the development of a high-throughput, colorimetric, continuous coupled enzyme assay for the screening of DAAT mutant libraries that is based on the use of D-amino acid oxidase (DAAO). In this assay, the D-amino acid product of DAAT is oxidized by DAAO with concomitant release of hydrogen peroxide, which is detected colorimetrically by the addition of horseradish peroxidase and D-dianisidine. Using this assay, we measured apparent K-M and k(cat) values for DAAT and identified mutants displaying altered substrate specificity via the screening of cell lysates in 96-well plates. The DAAO coupled assay is sensitive in that it allowed the detection of a DAAT mutant displaying an approximately 2000-fold decrease in k(cat)/K-M relative to wild type. In addition, the DAAO assay enabled the identification of two DAAT mutants (V33Y and V33G) that are more efficient than wild type at transaminating the non-native acceptor phenylpyruvate. We expect that this assay will be useful for the engineering of additional mutants displaying increased activity toward non-native substrates. (C) 2014 Elsevier Inc. All rights reserved.