Enzyme Reactor Using Enzyme-Coenzyme Conjugate

制备了葡萄糖脱氢酶-聚乙二醇-NAD偶联物（GlcDH-PEG-NAD），并研究了其作为NADH还原单元的动力学性质。以丙酮酸为原料，在含有GlcDH-PEG-NAD和乳酸脱氢酶（LDH）的PM10超滤膜反应器中连续生产L-乳酸。GlcDH-PEG-NAD作为大分子尺寸的NADH再生单元被证明适用于连续酶反应。然后，我们通过共价连接5-乙基吩嗪制备了以下酶-辅因子缀合物（也是NAD）到各种酶：共价连接的5-乙基吩嗪聚（乙二醇）-谷氨酸脱氢酶缀合物在一些实施方案中，所述缀合物包括共价连接的5-乙基吩嗪-葡萄糖脱氢酶-NAD缀合物（EP-PEG-GltDH）、共价连接的5-乙基吩嗪-葡萄糖脱氢酶-NAD缀合物（EP-GlcDH-NAD）和共价连接的5-乙基吩嗪-乳酸脱氢酶-NAD缀合物（EP-LDH-NAD）。这些偶联物显示出新的催化活性，EP部分作为人工催化基团用于催化 ...更多信息 在新的催化反应中，NADH（或其部分）被氧或四唑盐（如MTT）氧化，这些偶联物是独特的半合成酶，为人工设计酶提供了动力学基础。EP-PEG-GltDH作为NADH氧化酶起作用，GltDH部分的辅酶结合位点用作底物结合位点。NADH-氧化酶活性的动力学分析显示了EP部分的催化基团附近的底物结合位点的存在的影响。EP-GlcDH-NAD作为葡萄糖氧化酶起作用。这种活性是由于GlcDH和EP两个催化反应的耦合所致，动力学分析表明存在以下速率加速机制：高有效浓度、扩散催化反应的分子内耦合以及两种催化位点之间的多重连接。EP-LDH-NAD作为乳酸氧化酶起作用。动力学分析显示LDH部分的辅酶结合位点对NADH部分的氢化作用。少

Glucose-dehydrogenase-poly (ethylene glycol) -NAD conjugate (GlcDH-PEG-NAD) was prepared and its kinetic properties as an NADH-rageneration unit were investigated. L-Lactate was continuously produced from pyruvate in a reactor with a PM10 ultrafiltration membrane, and containing GlcDH-PEG-NAD and lactate dehydrogenase (LDH). GlcDH-PEG-NAD proved to be applicable in continuous enzyme reaction as an NADH-regeneration unit with a large molecular size.Then, we have prepared the following enzyme-cofactor conjugate by covalently linking 5-ethylphenazine (and also NAD) to various dehydrogenases : a covalently linked 5-ethylphenazinepoly (ethylene glycol) -glutamate dehydrogenases conjugate (EP-PEG-GltDH), a covalently linked 5-ethylphenazine-glucose dehydrogenase-NAD conjugate (EP-GlcDH-NAD), and a covalently linked 5-ethylphanazine-lactate dehydrogenase-NAD conjugate (EP-LDH-NAD). These conjugate show new catalytic activities, and the EP moiety works as an artificial catalytic group for the … More oxidation of NADH (or the NADH mojety) with oxygen or a tetrazolium salt such as MTT in the new catalytic reactions.These conjugates are unique semisynthetic enzymes, and provide us with kinetic basis for artificially designing enzymes. EP-PEG-GltDH works as an NADH oxidase, and the coenzyme-binding site of the GltDH moiety is used as a substrate-binding site. Kinetic analysis of the NADH-oxidase activity shows the effect of the presence of the substrate-binding site near the catalytic group of the EP moiety. EP-GlcDH-NAD works as a glucose oxidase. This activity is due to the coupling of the two catalytic reactions of the GlcDH and the EP moieties, and kinetic analysis shows the presence of the following rate accerelation mechanisms : high effective concentration, intramolecular coupling of succesive catalytic reactions, and multiple connection between the two kinds of the catalytic sites. EP-LDH-NAD works as a lactate oxidase. Kinetic analysis shows the effect of hyding the NADH moiety by the coenzyme-binding site of the LDH moiety. Less