Enzyme Reactor Using Enzyme-Coenzyme Conjugate

制备了葡萄糖-脱氢酶-聚乙二醇-NAD缀合物（GlcDH-PEG-NAD），并对其作为nadh生成单元的动力学性质进行了研究。以丙酮酸为原料，在PM10超滤膜反应器中连续制备l -乳酸，该反应器含有GlcDH-PEG-NAD和乳酸脱氢酶（LDH）。事实证明，GlcDH-PEG-NAD作为一种大分子大小的nadh再生单元适用于连续酶反应。然后，我们将5-乙基吩嗪（和NAD）与各种脱氢酶共价连接，制备了以下酶-辅因子偶联物：共价连接的5-乙基吩嗪聚（乙二醇）-谷氨酸脱氢酶偶联物（EP-PEG-GltDH），共价连接的5-乙基吩嗪-葡萄糖脱氢酶-NAD偶联物（EP-GlcDH-NAD）和共价连接的5-乙基吩嗪-乳酸脱氢酶-NAD偶联物（EP-LDH-NAD）。这些共轭物显示出新的催化活性，在新的催化反应中，EP部分作为人工催化基团对NADH（或NADH部分）与氧或四氮唑盐（如MTT）进行更多的氧化。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片段的辅酶结合位点被用作底物结合位点。nadh氧化酶活性的动力学分析表明，底物结合位点的存在对EP部分催化基团附近的影响。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作用，动力学分析表明存在以下速率加速机制：高效浓度、连续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是一种乳酸氧化酶。动力学分析表明，通过LDH片段的辅酶结合位点将NADH片段结合。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