NAD +/NADH recycling by coimmobilized lactate dehydrogenase and glutamate dehydrogenase

• Published in: Enzyme and microbial technology 1998-07, Vol.23 (1), p.49-57
• Main Authors: Le, Min, Means, Gary E.
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Biotechnology; Coimmobilization; controlled enzyme distribution; Fundamental and applied biological sciences. Psychology; Immobilization techniques; Methods. Procedures. Technologies; Multienzyme coimmobilization; NAD +/NADH recycling
• ISSN: 0141-0229; 1879-0909
• DOI: 10.1016/S0141-0229(98)00010-6

Most biologically active proteins bind irreversibly to Amberlite XAD-7 in dilute buffer at approximately neutral pH. Small amounts of protein (i.e., ∼4% w/w or less) are adsorbed at the periphery of the porous, spherical, beads where the protein is relatively accessible to external solvent and solvent components. Additional amounts of the same or different proteins up to ∼35% (w/w) absorb to deeper layers of the same beads. The order in which two or more proteins are adsorbed can be used to arrange them in a series of concentric layers on Amberlite XAD-7. An NAD +/NADH recycling system for the synthesis of l-glutamate from ammonia, α-ketoglutarate, and lactate was prepared by coimmobilizing rabbit muscle lactate dehydrogenase (LDH) and bovine liver glutamate dehydrogenase (GDH) in different amounts and in different arrangements on Amberlite XAD-7. The overall efficiency of that immobilized system based on the rates of glutamate production varied with both the ratio of the two enzymes and their arrangement on the polymer beads. Those variations suggest that NADH formation was rate limiting under most conditions and demonstrated the importance of enzyme proximity. The immobilized two-enzyme system, reflecting the pH dependence of its constituent enzymes, had optimal activity at pH ∼8.5 and retained up to 75% of its activity after one week of repeated use.