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首页> 外文期刊>Combinatorial Chemistry _High Throughput Screening >A Filter Paper-Based Assay for Laboratory Evolution of Hydrolases and Dehydrogenases
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A Filter Paper-Based Assay for Laboratory Evolution of Hydrolases and Dehydrogenases

机译：基于滤纸的实验室测定水解酶和脱氢酶的分析

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摘要

Industrially important enzyme classes such as hydrolases and dehydrogenases are often not amenable to laboratory evolution methods due to a lack of sensitive and reliable high-throughput screening (HTS) systems. We developed a conceptually novel and technically simple high-throughput screening system based on detection of volatile aldehydes with the sensitive reagent Purpald (4-amino-3-hydrazino-5-mercapto-1,2,4-triazole). The aldehyde detection takes place on a filter-paper that is pre-soaked with Purpald and covers the microtiter plate. The filter paper-based Purpald assay separates aldehyde detection from biocatalytical conversion and thereby avoids interferences from biological materials with assay components. This screening principle allows, to our knowledge, for the first time to determine the synthetic activity of hydrolases such as lipases and esterases in organic solvents in a 96-well whole-cell format. Its simplicity and cost-effectiveness make the reported HTS system suitable as fast pre-screen in laboratory evolution experiments and for semi-quantitative assays of improved mutants.
机译：由于缺乏灵敏和可靠的高通量筛选（HTS）系统，工业上重要的酶类别（例如水解酶和脱氢酶）通常不适合实验室进化方法。我们基于灵敏试剂Purpald（4-氨基-3-肼基-5-巯基-1,2,4-三唑）对挥发性醛的检测，开发了一种概念新颖，技术上简单的高通量筛选系统。醛的检测在预先用Purpald浸透并覆盖微量滴定板的滤纸上进行。基于滤纸的Purpald分析将醛的检测与生物催化转化分离开，从而避免了生物材料对分析组分的干扰。据我们所知，这种筛选原理首次确定了96孔全细胞形式的水解酶（如脂肪酶和酯酶）在有机溶剂中的合成活性。它的简单性和成本效益使所报道的HTS系统适合在实验室进化实验和改良突变体的半定量测定中进行快速预筛选。

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