Semi-rational engineering of an amino acid racemase that is stabilized in aqueous/organic solvent mixtures.,Biotechnology and Bioengineering

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Semi-rational engineering of an amino acid racemase that is stabilized in aqueous/organic solvent mixtures.
Biotechnology and Bioengineering ( IF 3.5 ) Pub Date : 2020-06-03 , DOI: 10.1002/bit.27449
Christian Femmer ₁ , Matthias Bechtold ₁ , Sven Panke ₁

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Enzymes are industrially applied under increasingly diverse environmental conditions that are dictated by the efforts to optimize overall process efficiency. Engineering the operational stability of biocatalysts to enhance their half‐lives under the desired process conditions is a widely applied strategy to reduce costs. Here, we present a simple method to enhance enzyme stability in the presence of monophasic aqueous/organic solvent mixtures based on the concept of strengthening the enzyme's surface hydrogen‐bond network by exchanging surface‐located amino acid residues for arginine. Suitable residues are identified from sequence comparisons with homologous enzymes from thermophilic organisms and combined using a shuffling approach to obtain an enzyme variant with increased stability in monophasic aqueous/organic solvent mixtures. With this approach, we increase the stability of the broad‐spectrum amino acid racemase of Pseudomonas putida DSM 3263 eightfold in mixtures with 40% methanol and sixfold in mixtures with 30% acetonitrile.

中文翻译：

在水/有机溶剂混合物中稳定的氨基酸消旋酶的半合理工程。

酶在工业上应用在日益多样化的环境条件下，这取决于优化整体过程效率的努力。设计生物催化剂的操作稳定性以提高其在所需工艺条件下的半衰期是一种广泛应用的降低成本的策略。在这里，我们基于通过将位于表面的氨基酸残基交换为精氨酸来加强酶的表面氢键网络的概念，提出了一种在单相水/有机溶剂混合物存在下提高酶稳定性的简单方法。通过与来自嗜热生物的同源酶的序列比较确定合适的残基，并使用改组方法组合以获得在单相水/有机溶剂混合物中稳定性增加的酶变体。恶臭假单胞菌DSM 3263 在与 40% 甲醇的混合物中是八倍，在与 30% 乙腈的混合物中是六倍。

更新日期：2020-06-03

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