Background

Glucaric acid, one of the aldaric acids, has been declared a “top value-added chemical from biomass”, and is especially important in the food and pharmaceutical industries. Biocatalytic production of glucaric acid from glucuronic acid is more environmentally friendly, efficient and economical than chemical synthesis. Uronate dehydrogenases (UDHs) are the key enzymes for the preparation of glucaric acid in this way, but the poor thermostability and low activity of UDH limit its industrial application. Therefore, improving the thermostability and activity of UDH, for example by semi-rational design, is a major research goal.

Results

In the present work, three UDHs were obtained from different Agrobacterium tumefaciens strains. The three UDHs have an approximate molecular weight of 32 kDa and all contain typically conserved UDH motifs. All three UDHs showed optimal activity within a pH range of 6.0–8.5 and at a temperature of 30 °C, but the UDH from A. tumefaciens (At) LBA4404 had a better catalytic efficiency than the other two UDHs (800 vs 600 and 530 s⁻¹ mM⁻¹). To further boost the catalytic performance of the UDH from AtLBA4404, site-directed mutagenesis based on semi-rational design was carried out. An A39P/H99Y/H234K triple mutant showed a 400-fold improvement in half-life at 59 °C, a 5 °C improvement in \( {\text{T}}_{ 5 0}^{ 1 0} \) value and a 2.5-fold improvement in specific activity at 30 °C compared to wild-type UDH.

Conclusions

In this study, we successfully obtained a triple mutant (A39P/H99Y/H234K) with simultaneously enhanced activity and thermostability, which provides a novel alternative for the industrial production of glucaric acid from glucuronic acid.

中文翻译：

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通过半理性工程提高根癌农杆菌LBA4404的尿酸盐脱氢酶的热稳定性和活性

背景

葡糖二酸是一种醛糖酸，已被宣布为“来自生物质的高附加值化学品”，在食品和制药行业中尤其重要。由葡糖醛酸生物催化生产葡糖二酸比化学合成更环保，有效和经济。铀酸脱氢酶（UDHs）是以此方式制备葡糖二酸的关键酶，但是UDH的热稳定性差和活性低限制了其工业应用。因此，例如通过半理性设计来提高UDH的热稳定性和活性是主要的研究目标。

结果

在目前的工作中，从不同的根癌农杆菌菌株中获得了三个UDH 。这三个UDH的分子量约为32 kDa，均包含通常保守的UDH基序。这三种UDH均在pH值为6.0-8.5且温度为30°C的条件下均表现出最佳活性，但是根癌农杆菌（At）LBA4404的UDH具有比其他两种UDH更好的催化效率（800 vs 600和530） s ^-1 mM ^-1）。为了进一步提高AtLBA4404对UDH的催化性能，进行了基于半合理设计的定点诱变。一个A39P / H99Y / H234K三重突变体在59°C时的半衰期提高了400倍，\（{\ text {T}} _ {5 0} ^ {1 0} \）则提高了5°C 。 与野生型UDH相比，其在30°C下的比值和比活度提高了2.5倍。

结论

在这项研究中，我们成功地获得了同时具有增强的活性和热稳定性的三重突变体（A39P / H99Y / H234K），这为从葡萄糖醛酸工业生产葡糖二酸提供了新的选择。