Abstract Chromohalobacter salixigens contains a uronate dehydrogenase termed CsUDH that can convert uronic acids to their corresponding C1,C6-dicarboxy aldaric acids, an important enzyme reaction applicable for biotechnological use of sugar acids. To increase the thermal stability of this enzyme for biotechnological processes, directed evolution using gene family shuffling was applied, and the hits selected from 2-tier screening of a shuffled gene family library contained in total 16 mutations, only some of which when examined individually appreciably increased thermal stability. Most mutations, while having minimal or no effect on thermal stability when tested in isolation, were found to exhibit synergy when combined; CsUDH-inc containing all 16 mutations had ΔK t0.5 +18 °C, such that kcat was unaffected by incubation for 1 h at ∼70 °C. X-ray crystal structure of CsUDH-inc showed tight packing of the mutated residue side-chains, and comparison of rescaled B-values showed no obvious differences between wild type and mutant structures. Activity of CsUDH-inc was severely depressed on glucuronic and galacturonic acids. Combining select combinations of only three mutations resulted in good or comparable activity on these uronic acids, while maintaining some improved thermostability with ΔK t0.5 ∼+ 10 °C, indicating potential to further thermally optimize CsUDH for hyperthermophilic reaction environments.

中文翻译：

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Chromohalobacter salixigens 糖醛酸脱氢酶：定向进化以提高热稳定性和突变体 CsUDH-inc X 射线晶体结构

摘要 Chromohalobacter salixigens 含有一种称为 CsUDH 的糖醛酸脱氢酶，可将糖醛酸转化为相应的 C1、C6-二羧基醛糖酸，这是一种适用于糖酸生物技术应用的重要酶反应。为了提高这种酶在生物技术过程中的热稳定性，应用了使用基因家族改组的定向进化，并且从包含总共 16 个突变的改组基因家族文库的 2 层筛选中选择的命中，其中只有一些在单独检查时明显提高热稳定性。大多数突变，虽然在单独测试时对热稳定性的影响很小或没有影响，但被发现在组合时表现出协同作用；包含所有 16 个突变的 CsUDH-inc 具有 ΔK t0.5 +18 °C，因此 kcat 不受在~70 °C 下孵育 1 小时的影响。CsUDH-inc 的 X 射线晶体结构显示突变残基侧链紧密堆积，重新调整的 B 值比较显示野生型和突变体结构之间没有明显差异。CsUDH-inc 的活性在葡萄糖醛酸和半乳糖醛酸上受到严重抑制。将仅三个突变的选择组合组合导致对这些糖醛酸具有良好或相当的活性，同时保持 ΔK t0.5 ∼ + 10 °C 的一些改进的热稳定性，表明有可能进一步热优化 CsUDH 以适应超嗜热反应环境。