A fast and sensitive assay for measuring the activity and enantioselectivity of transaminases
A fast and sensitive assay for measuring the activity and enantioselectivity of transaminases
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DOI：
10.1039/c0cc02919j
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发表时间：
2011-01-01
影响因子：
4.9
通讯作者：
Lloyd, Richard C.
中科院分区：
文献类型：
--
作者：
Hopwood, Jennifer;Truppo, Matthew D.;Lloyd, Richard C.
氨基转移酶(ATA)是一种依赖于磷酸吡哆醛的酶，催化氨从胺供体到酮受体的可逆转移。最近，它们作为合成手性纯手性伯胺的生物催化剂引起了人们的极大兴趣。(1)特别关注(I)克服底物/产物抑制的问题，(Ii)解决与酮到胺的不平衡转化有关的问题，以及(Iii)开发具有更广泛底物专一性的ATAs。(2)同样重要的是能够从活性和对映体选择性两方面快速筛选新底物的ATAs。以前筛选ATA活性的方法是基于使用指示剂苯基红监测pH变化，(3)有色铜-丙氨酸络合物的形成(4)以及一种优雅的紫外分光光度方法。(5)在这里，我们描述了一种简单的基于比色法的方法，并证明它可以用于测定广泛范围的ATA底物的活性和对映体选择性。方案1概述了新筛查方法的基础。胺1用ATA和丙酮酸钠2处理。成功的底物导致丙酮酸钠的转化
Amino transaminases (ATAs) are pyridoxal phosphate dependent enzymes that catalyse the reversible transfer of ammonia from an amine donor to a ketone acceptor. Recently they have attracted considerable interest as biocatalysts for the synthesis of enantiomerically pure chiral primary amines.(1) Particular attention has been paid to (i) overcoming problems of substrate/product inhibition, (ii) solving issues related to poor equilibrium conversions of ketone to amine and (iii) developing ATAs with broader substrate specificity.(2) Equally important is the ability to be able to rapidly screen ATAs for new substrates, with respect to both activity and enantio-selectivity. Previous methods for screening for ATA activity have been based upon monitoring pH changes using the indicator dye phenyl red,(3) formation of coloured Cu-alanine complexes(4) and also an elegant UV spectrophotometric protocol.(5) Herein we describe a simple colorimetric-based method and show that it can be used to determine both the activity and enantioselectivity of a wide range of ATA substrates. Scheme 1 outlines the basis for the new screening method. Amines 1 are treated with an ATA and sodium pyruvate 2. Successful substrates result in conversion of sodium pyruvate