Enzyme Reactor Using Enzyme-Coenzyme Conjugate

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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