Abstract

NAD(P)⁺-dependent formate dehydrogenase (FDH, EC 1.2.1.2.) is actively used in processes of chiral synthesis by oxidoreductases with systems of reduced cofactor regeneration. The efficient use of FDH in such systems requires simple and fast enzyme purification. Metal-chelate affinity chromatography is widely used for such purposes. The method requires the presence of at least six His residues at N- or C-terminus of protein. The addition of extra His residues can affect enzyme properties. The computer modeling of the structure of FDH from bacterium Pseudomonas sp. 101 with different positions of His₆ sequence showed that the optimal case is His-tag at N-terminus. Three types of PseFDH with His₆ were prepared: wild-type NAD⁺-dependent enzyme and two mutant NADP⁺-specific forms. New PseFDHs were obtained as homogeneous preparations through a one-step purification procedure. The comparison of PseFDHs with and without His-tag showed that they have similar kinetic properties.

中文翻译：

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His 6序列对细菌假单胞菌门甲酸脱氢酶性质的影响。101

摘要

NAD（P）⁺依赖的甲酸脱氢酶（FDH，EC 1.2.1.2。）积极用于氧化还原酶与辅助因子还原系统的手性合成过程中。在此类系统中有效使用FDH需要简单，快速的酶纯化。金属螯合物亲和色谱法广泛用于这种目的。该方法需要在蛋白质的N或C末端存在至少六个His残基。添加额外的His残基会影响酶的性质。计算机模拟细菌假单胞菌FDH的结构。101个具有His ₆序列不同位置的101表明，最佳情况是N末端的His标签。制备了三种带有His ₆的PseFDH ：野生型NAD^+-依赖性酶和两种突变的NADP ⁺ -特异性形式。通过一步纯化程序，将新的PseFDHs作为均相制剂获得。具有和不具有His标签的PseFDHs的比较表明，它们具有相似的动力学性质。