Enzymatic hydration activity assessed by selective spectrophotometric detection of alcohols: A novel screening assay using oleate hydratase as a model enzyme
Enzymatic hydration activity assessed by selective spectrophotometric detection of alcohols: A novel screening assay using oleate hydratase as a model enzyme
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DOI：
10.1002/biot.201300412
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发表时间：
2014-06-01
影响因子：
4.7
通讯作者：
Otten, Linda G.
中科院分区：
文献类型：
--
作者：
Hiseni, Aida;Medici, Rosario;Otten, Linda G.
羟基脂肪酸是一种高附加值的化合物，广泛应用于聚合物、润滑剂、乳化剂和稳定剂中，具有潜在的药用价值。在自然界中，HFA由几种酶区域特异性合成，包括P450单加氧酶、脂氧合酶、水合酶、12-羟化酶和二醇脱氢酶。对HFA不断增长的需求保证了开发简单有效的分析方法，该方法能够在其不饱和前体存在的情况下高通量检测羟基化产物。本文描述了一种用于检测醇的新的高通量测定法，其使用来自脑膜炎败血症菌的油酸水合酶（OHase，EC 4.2.1.53）作为模型酶。开发的测定是基于选择性分光光度检测的烷基亚硝酸盐之间的羟基基团和亚硝酸反应形成的。该测定证明可以区分不饱和脂肪酸以及小的环状和非环状不饱和烯烃及其相应的醇。较低的检测限为1.5-3 mM，具有优异的Z '因子。使用OHase与油酸的酶促反应导致各种酶制剂的Z因子稍微较低。这种小规模的测定能够从突变体文库中快速发现新的微生物或改进的酶，并且将可用于涉及脂肪酸水合（去水合）酶的生物催化策略。
Hydroxy fatty acids (HFAs) are high-added-value compounds, which are incorporated in polymers, lubricants, emulsifiers and stabilizers and have potential medicinal use. In nature, HFAs are regio-specifically synthesized by several enzymes, including P450 monooxygenases, lipoxygenases, hydratases, 12-hydroxylases, and diol synthases. The growing demand for HFAs warrants the development of simple and efficient analytical methods that enable high-throughput detection of the hydroxylated product in the presence of its unsaturated precursor. Herein a novel high-throughput assay for the detection of alcohols is described using oleate hydratase (OHase, EC 4.2.1.53) from Elizabethkingia meningoseptica as the model enzyme. The developed assay is based on the selective spectrophotometric detection of alkyl nitrites formed upon the reaction between the hydroxyl group and nitrous acid. The assay proved to discriminate between unsaturated fatty acids as well as small cyclic and acyclic unsaturated alkenes and their corresponding alcohols. Lower detection limits were 1.5-3 mM with excellent Z'-factors. Enzymatic reactions using OHase with oleic acid resulted in somewhat lower Z-factors for various enzyme preparations. This small scale assay can enable fast discovery of new microorganisms or improved enzymes from mutant libraries and will be useful for biocatalytic strategies involving fatty acid (de)hydrating enzymes.