Biomolecular Engineering Lab, Dipartimento di Elettronica, Informazione e Bioingegneria, Politecnico di Milano, Piazza Leonardo da Vinci 32, 20133, Milano, Italy.
Center for Nano Science and Technology @Polimi, Istituto Italiano di Tecnologia, Via G. Pascoli 70/3, 20133, Milano, Italy.
Sci Rep. 2018 Feb 14;8(1):3042. doi: 10.1038/s41598-018-19991-x.
Amadoriases are a class of FAD-dependent enzymes that are found in fungi, yeast and bacteria and that are able to hydrolyze glycated amino acids, cleaving the sugar moiety from the amino acidic portion. So far, engineered Amadoriases have mostly found practical application in the measurement of the concentration of glycated albumin in blood samples. However, these engineered forms of Amadoriases show relatively low absolute activity and stability levels, which affect their conditions of use. Therefore, enzyme stabilization is desirable prior to function-altering molecular engineering. In this work, we describe a rational design strategy based on a computational screening method to evaluate a library of potentially stabilizing disulfide bonds. Our approach allowed the identification of two thermostable Amadoriase I mutants (SS03 and SS17) featuring a significantly higher T (55.3 °C and 60.6 °C, respectively) compared to the wild-type enzyme (52.4 °C). Moreover, SS17 shows clear hyperstabilization, with residual activity up to 95 °C, whereas the wild-type enzyme is fully inactive at 55 °C. Our computational screening method can therefore be considered as a promising approach to expedite the design of thermostable enzymes.
阿玛多里酶是一类依赖黄素腺嘌呤二核苷酸（FAD）的酶，存在于真菌、酵母和细菌中，能够水解糖化氨基酸，将糖部分从氨基酸部分中切断。到目前为止，经过工程改造的阿玛多里酶主要在测量血液样本中糖化白蛋白的浓度方面有实际应用。然而，这些经过工程改造的阿玛多里酶表现出相对较低的绝对活性和稳定性水平，这影响了它们的使用条件。因此，在进行功能改变的分子工程之前，需要对酶进行稳定化处理。在这项工作中，我们描述了一种基于计算筛选方法的合理设计策略，用于评估潜在稳定二硫键的文库。我们的方法能够识别出两个耐热性更高的阿玛多里酶 I 突变体（SS03 和 SS17），它们的 T（55.3°C 和 60.6°C）分别比野生型酶（52.4°C）高很多。此外，SS17 表现出明显的超稳定性，残余活性高达 95°C，而野生型酶在 55°C 时完全失活。因此，我们的计算筛选方法可以被认为是一种有前途的方法，可以加速耐热酶的设计。
