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

使用辅酶收集系统开发多酶生物反应器。

• 批准号: 07456051
• 负责人: OHTA Takahisa
• 金额: $ 5.06万
• 依托单位: Kogakuin University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07456051/
• 关键词: BIOREACTOR; IMMOBILIZED ENZYMES; COENZYMES; NAD^+; ATP; バイオイアクター; NAD

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.

一、辅酶收集柱系统建立了辅酶的最佳收集条件.最佳收集条件：吸附：50 mM Tris-HCl，pH8.5;洗脱：50 mM MES-NaOH，pH6.02。最佳收集条件：吸附：50 mM GlyGly-NaOH，pH9.5，洗脱：50 mM柠檬酸，pH4.03。吸附条件：50 mM CHS-NaOH，pH9.5，洗脱条件：50 mM柠檬酸盐，pH4.0 Ⅱ.辅酶消耗和再生模型体系我们利用AffiGE 115.1固定化酶，消耗和再生NADH或ATP。NAD^+/NADH反应体系乳酸脱氢酶（LDH）固定化条件：100 mMMOPS-NaOH，pH6.0甲酸脱氢酶（FDH）固定化条件：100 mMMOPS-NaOH，pH8.02。ATP/ADP反应体系己糖激酶（HK）固定条件：100 mM MOPS-NaOH，pH6.0丙酮酸激酶（PK）固定条件：100 mM 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储存过程中有所增加。