Roth Teresa, Beer Barbara, Pick André, Sieber Volker
Chair of Chemistry of Biogenic Resources, Straubing Centre of Science, Technical University of Munich, Schulgasse 16, 94315, Straubing, Germany.
Roche Diagnostics GmbH, Nonnenwald 2, 82377, Penzberg, Germany.
AMB Express. 2017 Dec;7(1):103. doi: 10.1186/s13568-017-0405-2. Epub 2017 May 23.
Aldaric acids represent biobased 'top value-added chemicals' that have the potential to substitute petroleum-derived chemicals. Until today they are mostly produced from corresponding aldoses using strong chemical oxidizing agents. An environmentally friendly and more selective process could be achieved by using natural resources such as seaweed or pectin as raw material. These contain large amounts of uronic acids as major constituents such as glucuronic acid and galacturonic acid which can be converted into the corresponding aldaric acids via an enzyme-based oxidation using uronate dehydrogenase (Udh). The Udh from Agrobacterium tumefaciens (UdhAt) features the highest catalytic efficiency of all characterized Udhs using glucuronic acid as substrate (829 s mM). Unfortunately, it suffers from poor thermostability. To overcome this limitation, we created more thermostable variants using semi-rational design. The amino acids for substitution were chosen according to the B factor in combination with four additional knowledge-based criteria. The triple variant A41P/H101Y/H236K showed higher kinetic and thermodynamic stability with a T value of 62.2 °C (3.2 °C improvement) and a ∆∆G of 2.3 kJ/mol compared to wild type. Interestingly, it was only obtained when including a neutral mutation in the combination.
醛糖二酸是具有替代石油衍生化学品潜力的生物基“高附加值化学品”。直到如今，它们大多是使用强化学氧化剂从相应的醛糖生产而来。通过使用海藻或果胶等自然资源作为原料，可以实现一种环境友好且更具选择性的工艺。这些资源含有大量的糖醛酸作为主要成分，如葡萄糖醛酸和半乳糖醛酸，它们可以通过使用糖醛酸脱氢酶（Udh）的基于酶的氧化反应转化为相应的醛糖二酸。来自根癌农杆菌的Udh（UdhAt）在所有已表征的以葡萄糖醛酸为底物的Udh中具有最高的催化效率（829 s mM）。不幸的是，它的热稳定性较差。为了克服这一限制，我们使用半理性设计创建了更具热稳定性的变体。根据B因子并结合另外四个基于知识的标准选择用于替换的氨基酸。与野生型相比，三重变体A41P/H101Y/H236K表现出更高的动力学和热力学稳定性，其T值为62.2°C（提高了3.2°C），∆∆G为2.3 kJ/mol。有趣的是，只有在组合中包含一个中性突变时才能获得该变体。
