TTC-based screening assay for ?-transaminases: a rapid method to detect reduction of 2-hydroxy ketones.
TTC-based screening assay for ?-transaminases: a rapid method to detect reduction of 2-hydroxy ketones.
复制标题
基于 TTC 的 β-转氨酶筛选测定：检测 2-羟基酮还原的快速方法。
DOI：
10.1016/j.jbiotec.2011.12.023
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发表时间：
2012
影响因子：
4.1
通讯作者：
Sehl T
中科院分区：
文献类型：
--
作者：
Sehl T
开发了一种用于ω-转氨酶的基于TTC的快速筛选测定，以确定具有2-羟基酮基序的底物的转化。化合物在氯化2，3，5-三苯基四唑鎓（TTC）存在下的氧化导致无色TTC还原为红色的1，3，5-三苯基甲瓒。各种脂肪族、芳香族-芳香族和芳香族-芳香族2-羟基酮的酶促还原胺化可通过底物消耗导致的红色减少来测定。转化可以基于反应在510 nm下通过荧光定量法定量，例如在96孔板中使用粗细胞提取物。由于该测定不依赖于不同胺供体的选择，因此筛选了一组ω-转氨酶以检测2-羟基酮与三种不同胺供体的转化：l-丙氨酸、（S）-α-甲基苄胺和苄胺。结果可以使用HPLC和GC分析进行验证，显示偏差仅为5- 10%。使用这种方法，酶被鉴定，证明乙偶姻和苯乙酰基甲醇向相应胺的高转化率。在这些酶中，已经鉴定了三种新的野生型ω-转氨酶。
A rapid TTC-based screening assay for ω-transaminases was developed to determine the conversion of substrates with a 2-hydroxy ketone motif. Oxidation of the compounds in the presence of 2,3,5-triphenyltetrazolium chloride (TTC) results in a reduction of the colourless TTC to a red-coloured 1,3,5-triphenylformazan. The enzymatic reductive amination of a wide range of various aliphatic, aliphatic–aromatic and aromatic–aromatic 2-hydroxy ketones can be determined by the decrease of the red colouration due to substrate consumption. The conversion can be quantified spectrophotometrically at 510nm based on reactions, e.g. with crude cell extracts in 96-well plates. Since the assay is independent of the choice of diverse amine donors a panel of ω-transaminases was screened to detect conversion of 2-hydroxy ketones with three different amine donors: l-alanine, (S)-α-methylbenzylamine and benzylamine. The results could be validated using HPLC and GC analyses, showing a deviation of only 5–10%. Using this approach enzymes were identified demonstrating high conversions of acetoin and phenylacetylcarbinol to the corresponding amines. Among these enzymes three novel wild-type ω-transaminases have been identified.