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

1、辅酶收集柱系统：建立了收集辅酶的最佳条件。NAD^+或nadhad的最佳吸附条件：50mM - Tris-HCl， pH8.5，洗脱：50mM - MES-NaOH，pH6.02。吸附：50mM GlyGly-NaOH，pH9.5，洗脱：50mM柠檬酸盐，pH4.03。吸附条件：50mM CHS-NaOH，pH9.5，洗脱条件：50mM柠檬酸盐，pH4.0II。辅酶消耗和再生的模型系统。我们用AffiGE115.1固定酶来消耗和再生NADH或ATP。NAD^+/ nadhiml -乳酸脱氢酶（LDH）的固定化条件：100mmmps - naoh， ph6.0甲酸脱氢酶（FDH）的固定化条件：100mM mps - naoh，pH8.02。丙酮酸激酶（PK）固定化条件：100mM HEPES，pH8.03。固定化酶的稳定性：固定化FDH在一个月后仍保持80%的活性。其他固定化酶的活性在贮藏过程中有所增加。

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.