Site-to-site directed immobilization of enzymes with bis-NAD analogues.

Abstract

Lactate dehydrogenase (L-lactate:NAD' oxidoreductase, EC 1.1.1.27) and alcohol dehydrogenase (alcohol: NAD' oxidoreductase, EC 1.1.1.1) have been crosslinked with glutaraldehyde on agarose beads. The crosslinking was performed while the two enzymes were spatially arranged with their active sites facing one another with the aid of a bis-NAD analogue. Subsequently the bis-NAD analogue was allowed to diffuse out. By using a third enzyme, lipoamide dehydrogenase (NADH:lipoamide oxidoreductase, EC 1.6.4.3), which was also coupled to the same beads and which competes with lactate dehydrogenase for the NADH produced by alcohol dehydrogenase, the effect of site-tosite directed immobilization was studied. It was found that much more NADH than was theoretically expected (50% instead of 19% of produced NADH) was oxidized by lactate dehydrogenase, which indicates that the NADH was preferentially channeled to lactate dehydrogenase due to the juxtapositioned active sites of the two enzymes.Several coimmobilized multistep enzyme systems have been described in the literature (1-6). For example, an immobilized system composed of the sequence malate dehydrogenase/citrate synthase showed a consistantly higher overall steady-state rate (6). More recently, a four-enzyme sequence (7) and even the enzymes of a complete metabolic cycle, the urea cycle, have been coimmobilized to supports (8). In the latter case, the immobilized cyclic enzyme system again was more efficient than the corresponding soluble system. These effects have been partly attributed to the close proximity of the enzymes and partly to the diffusional restrictions imposed by the Nernst unstirred layer around the enzymes (2, 9).

Multienzymatic Cascade Reactions via Enzyme Complex by Immobilization

“…By mimicking cascade enzymatic pathways, co-immobilization of multienzymes can lead to benefits such as lower costs of operation, separation, time, and waste treatment (Figure c). , Furthermore, coupling several steps together can drive the process toward the desired equilibrium reaction . During co-immobilization, each enzyme can be randomly distributed, , assembled at specific positions, , or formed into a localized compartment, as shown in Figure . − As a result, each enzyme can be associated with regulated activity leading to substrate channeling. These specific assemblies of multienzyme complexes have a major advantage.…”

Pluronic-Based Nanocarrier Platform Encapsulating Two Enzymes for Cascade Reactions

“…Since biosynthesis of metabolites and commercial synthesis of fine chemicals usually requires multiple reaction steps, − cascade reactions involving multiple enzymes attract more attention these days . Many enzyme pairs have been studied for cascade reactions. − Coimmobilization of enzymes is particularly useful in certain situations. First, in cascade reactions, the efficiency of one of the enzymes can be enhanced by in situ supplementation of its substrate. − Second, the coimmobilized enzymes can carry out cascade reactions as a one-pot reaction in contrast to conventional multistep processes.…”

Interenzyme Substrate Diffusion for an Enzyme Cascade Organized on Spatially Addressable DNA Nanostructures

“…In the future, it will be important to study the effect of enzyme orientation on the activity of assembled enzyme complexes as well. 15 With the further development of DNA-protein attachment chemistry through site-specific conjugation or ligand capture, 16 it should be possible to start to direct the flow of substrate molecules between active sites using some of the concepts and tools discussed above.…”