Reaction mechanism and application of NAD-binding dehydrogenases

1)甲醛歧化酶的纯化及性质。从恶臭假单胞菌F61中分离纯化了结晶型甲醛歧化酶。该酶由两个相同的亚基组成，Mr为44000。每个亚基含有1摩尔NAD(H)和2摩尔锌/摩尔。2)稳态动力学研究表明，NAD(H)在每个活性中心与NAD(H)牢固结合(但不是共价结合)，不被外源NAD(H)或类似物取代。尽管该酶的底物专一性与传统的NAD连接的乙醇脱氢酶相似，但该酶应根据整个反应将其归类为乙醇：乙醛氧化还原酶(EC 1.1.99.X)。3)固定化FDM并转化为甲醇生成甲酸盐。通过在氨基甲酸酯预聚体(PU-6)中的固定化，可以极大地稳定FDM值。甲醇转化为甲酸盐的反应是在由乙醇氧化酶组成的固定化酶系统中进行的。用多形汉逊酵母和恶臭汉逊酵母的完整细胞混合物。此外，固定化大大提高了细胞混合物的稳定性，周期添加的600 mM甲醇在12h内转化为甲酸盐，产率为75%。固定化细胞系统还能有效地将几种脂肪醇从C_1到C_4转化为相应的酸。4)NAD结合甲酸脱氢酶的利用。通过有机合成的方法将NAD共价结合到甲酸脱氢酶上。甲酸脱氢异构酶反应。其中，在含有亮氨酸脱氢酶、甲酸盐-NAD络合物和底物的反应体系中，2-氧代异己酸的酶还原反应按化学计量比进行。

1) Purification and properties of formaldehyde dismutase. Formaldehyde dismutase (FDM) was purified in crystalline form from Pseudomonas putida F61. The enzyme is composed of our identical subunits with a Mr of 44000. Each subunit contains 1 mol NAD(H) and 2 mol zinc/mol.2) The steady-state kinetics of FDM indicates that NAD(H) binds firmly (but not covalently) at each active site, and is not replaced by exogenous NAD(H) nor the analogues. Although the substrate specificity of the enzyme seems to be similar to those of conventional NAD-linked alcohol dehydrogenase, the enzyme should be classified a novel enzyme, alcohol:aldehyde oxidoreductase (EC 1.1.99.X) on the basis of the overall reaction.3) Immobilization of FDM and conversion to methanol to formate. FDM was greatly stabilized by immobilization in a urethane prepolymer (PU-6). Conversion of methanol to formate occurred in a reaction with an immobilized enzyme system consisting of alcohol oxidase. catalase and FDM, and with an intact cell-mixture of Hansenula polymorpha and P. putida. Furthermore, the stability of the cell-mixture was greatly improved by the immobilization, the 600 mM methanol added periodically being converted to formate in a 75% yield in 12h. The immobilized cell-system was also effective for the conversion of several aliphatic alcohols, C_1 to C_4, to the corresponding acids.4) Utilization of NAD-binding formate dehydrogenase. NAD was covalently bound to formate dehydrogenase by a method of organic synthesis. The formate dehydrogenasegenase reactions. Among them, the enzymatic reduction of 2-oxoisocaproate to leucine was occurred stoichiometrically in the reaction system containing leucine dehydrogenase, formate-NAD complex and substrates.