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首页> 外文期刊>Journal of Biotechnology >A novel screening assay for hydroxynitrile lyases suitable for high-throughput screening
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A novel screening assay for hydroxynitrile lyases suitable for high-throughput screening

机译：一种适用于高通量筛选的羟腈裂解酶的新型筛选方法

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摘要

Hydroxynitrile lyases (Hnls) are important biocatalysts for the synthesis of optically pure cyanohydrins, which are used as precursors and building blocks for a wide range of high price fine chemicals. Although two Hnl enzymes, from the tropical rubber tree Hevea brasiliensis and from the almond tree Prunus amygdalus, are already used for large scale industrial applications, the enzymes still need to be improved and adapted to the special demands of industrial processes. In many cases directed evolution has been the method of choice to improve enzymes, which are applied as industrial biocatalysts. The screening procedure is the most crucial point in every directed evolution experiment. Herein, we describe the successful development of a novel screening assay for Hnls and its application in high-throughput screening of Escherichia coli mutant libraries. The new assay allows rapid screening of mutant libraries and facilitates the discovery of improved enzyme variants. Hnls catalyze the cleavage of cyanohydrins to hydrocyanic acid and the corresponding aldehyde or ketone. The enzyme assay is based on the detection of hydrocyanic acid produced, making it an all-purpose screening assay, without restriction to any kind of substrate. The gaseous HCN liberated within the Hnl reaction is detected by a visible colorimetric reaction. The facile, highly sensitive and reproducible screening method was validated by identifying new enzyme variants with novel substrate specificities.
机译：羟基腈裂解酶（Hnls）是合成光学纯氰醇的重要生物催化剂，它们可用作各种高价精细化学品的前体和基石。尽管来自热带橡胶树巴西橡胶树和杏仁树杏树的两种Hnl酶已被用于大规模工业应用，但仍需要改进并适应工业过程的特殊要求。在许多情况下，定向进化已成为改善酶的一种选择方法，这些酶被用作工业生物催化剂。筛选程序是每个定向进化实验中最关键的一点。在这里，我们描述了Hnls的新型筛选测定法的成功开发及其在大肠杆菌突变体文库的高通量筛选中的应用。新的测定方法可以快速筛选突变体文库，并有助于发现改良的酶变体。 Hnls催化氰醇裂解为氢氰酸和相应的醛或酮。酶测定法基于对产生的氢氰酸的检测，使其成为一种通用的筛选测定法，而不受限于任何种类的底物。通过可见比色反应检测在Hn1反应中释放的气态HCN。通过鉴定具有新型底物特异性的新酶变体，验证了简便，高度敏感和可重现的筛选方法。

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