Development of Multi-enzyme bioreactors using coenzyme-collecting system.

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I.Coenzyme collecting column systemWe extablished optimal conditions for collecting coenzymes.1. Optimal conditons for collecting NAD^+ or NADHAdsorption : 50mM Tris-HCl, pH8.5, Elution : 50mM MES-NaOH,pH6.02. Optimal conditions for collecting ATP or AMPAdsorption : 50mM GlyGly-NaOH,pH9.5, Elution : 50mM Citrate, pH4.03. Optimal conditions for collecting ADPAdsorption : 50mM CHS-NaOH,pH9.5, Elution : 50mM Citrate, pH4.0II.Model systems for consumption and regeneration of coenzymes.We immobilized enzymes for consuming and regenerating NADH or ATP using AffiGE115.1. Reaction system for NAD^+/NADHImmobilizing condition for L-lactate dehydrogenase (LDH) : 100mMMOPS-NaOH,pH6.0Immobilizing condition for formate dehydrogenase (FDH) : 100mM MOPS-NaOH,pH8.02. Reaction system for ATP/ADPImmobilizing condition for hexokinase (HK) : 100mM MOPS-NaOH,pH6.0Immobilizing condition for pyruvate kinase (PK) : 100mM HEPES,pH8.03. Stability of immobilized enzymesImmobilized FDH had kept 80% of its activity after one month. Activities of other immobilized enzymes somewhat increased during storage.