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

• Project/Area Number: 07456051
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: 応用微生物学・応用生物化学
• Research Institution: Kogakuin University
• Principal Investigator: OHTA Takahisa Kogakuin University, Department of Applied Chemistry Professor, 工学部, 教授 (30011844)
• Co-Investigator(Kenkyū-buntansha): SAKAGUCHI Masayoshi Kogakuin University, Department of Applied Chemistry Assistant, 工学部, 助手 (80281351)
• Project Period (FY): 1995 – 1996
• Project Status: Completed (Fiscal Year 1996)
• Budget Amount: ¥7,900,000 (Direct Cost: ¥7,900,000); Fiscal Year 1996: ¥800,000 (Direct Cost: ¥800,000); Fiscal Year 1995: ¥7,100,000 (Direct Cost: ¥7,100,000)
• Keywords: BIOREACTOR / IMMOBILIZED ENZYMES / COENZYMES / NAD^+ / ATP / バイオイアクター / NAD

Research Abstract

I.Coenzyme collecting column system
We extablished optimal conditions for collecting coenzymes.
1. Optimal conditons for collecting NAD^+ or NADH
Adsorption : 50mM Tris-HCl, pH8.5, Elution : 50mM MES-NaOH,pH6.0
2. Optimal conditions for collecting ATP or AMP
Adsorption : 50mM GlyGly-NaOH,pH9.5, Elution : 50mM Citrate, pH4.0
3. Optimal conditions for collecting ADP
Adsorption : 50mM CHS-NaOH,pH9.5, Elution : 50mM Citrate, pH4.0
II.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^+/NADH
Immobilizing condition for L-lactate dehydrogenase (LDH) : 100mMMOPS-NaOH,pH6.0
Immobilizing condition for formate dehydrogenase (FDH) : 100mM MOPS-NaOH,pH8.0
2. Reaction system for ATP/ADP
Immobilizing condition for hexokinase (HK) : 100mM MOPS-NaOH,pH6.0
Immobilizing condition for pyruvate kinase (PK) : 100mM HEPES,pH8.0
3. Stability of immobilized enzymes
Immobilized FDH had kept 80% of its activity after one month. Activities of other immobilized enzymes somewhat increased during storage.