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

“…50,51 Furthermore, coupling several steps together can drive the process toward the desired equilibrium reaction. 52 During co-immobilization, each enzyme can be randomly distributed, 98,99 assembled at specific positions, 100,101 or formed into a localized compartment, as shown in Figure 4. 102−105 As a result, each enzyme can be associated with regulated activity leading to substrate channeling.…”

Characterization of a Bifunctional Enzyme Fusion of Trehalose-6-Phosphate Synthetase and Trehalose-6-Phosphate Phosphatase of Escherichia coli

“…We assumed that the physical proximity of two enzymes catalyzing sequential reactions might increase the reaction rate by facilitating transfer of the reaction intermediate when they are present in a complex. A variety of techniques have been applied to better understand the proximity effect of enzymes catalyzing sequential reactions, including cross-linking and coimmobilization (23,32,34). In many of these cases, the organized enzyme systems exhibited different kinetic or catalytic properties compared with their free counterparts in bulk solution.…”

Production of biofuels and biochemicals by in vitro synthetic biosystems: Opportunities and challenges

“…Enzyme components can be randomly distributed (Betancor et al, 2006;El-Zahab et al, 2004), positionally assembled (El-Zahab et al, 2004), and even the active site of an enzyme face to the one of another enzyme (Mansson et al, 1983) on solid supports. The enhanced reaction rates among co-immobilization of cascade enzymes have been observed for several systems (Mateo et al, 2006;Zhang, 2011b), but direct cross linking could lead to loss of enzyme activity (Sheldon and van Pelt, 2013).…”