## Results
### Coupling MDH\({}^{*}\) and GDH for Cofactor Recycling  
Having identified buffer conditions that could support the activity of both enzymes, the activities and cofactor recycling capabilities of purified MDH\({}^{*}\) and GDH were assayed within the same experiment at the optimal conditions of 65\({}^{\circ}\)C, 5.6% O\({}_{2}\) and 12.5% CO\({}_{2}\). Pyruvate and glucose concentrations were each adjusted to 30 mM, the concentration of HEPES was kept at 500 mM, and all other components were added to the reaction as shown in Table 1. Two different experimental setups were used to determine the success of coupling the two enzymes. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Experiment** & **O\({}_{2}\) (\%)** & **CO\({}_{2}\) (\%)** & **Temperature** & **Initial Rate** & **Initial Rate \({}^{*}\)** \\
**Number** & **O\({}_{2}\)** & **CO\({}_{2}\)** & **(\({}^{\circ}\)C)** & **(mM/min)** & **(mM/min)** \\ \hline
1 & 2.5 & 5 & 55 & 0.068 & 0.066 \\ \hline
2 & 7.5 & 5 & 55 & 0.061 & 0.064 \\ \hline
3 & 2. In the first setup, changes in pyruvate concentration were examined, which would indicate the conversion of NADH to NAD\({}^{+}\) and pyruvate to malate by MDH\({}^{\star}\). The reaction was tested under optimal conditions (65 \({}^{\circ}\)C, 5.6% O\({}_{2}\) and 12.5% CO\({}_{2}\)), by the addition of NADH. In the second setup, the same reaction mixture was used but the reaction was initiated by the addition of NAD\({}^{+}\) instead of NADH. Changes in the NADH concentration were monitored for 2 h with a plate reader. The final NADH concentrations were compared with an enzyme-free reaction control containing the same initial concentration of NADH as the first experimental setup (Table 3). Despite the presence of 30 mM pyruvate, GDH still displayed activity. The NADH consumption in the first setup demonstrated the activity of MDH\({}^{\star}\). The concentrations of malate and gluconate produced by the cascade were also established by deproteinizing samples of the reaction mixture, diluting them 10-fold, and analyzing them via HPIC. However, due to the high dilution factor, malate peaks were below the detection limit and pyruvate as the substrate was in excess; hence, small changes in concentrations were indistinguishable. However, gluconic acid production was detected in both cases, demonstrating that cofactor recycling had been achieved. To detect malate by HPIC, 2 mL cascade reactions were set up and monitored via plate reader, comprising 13.2U GDH and 3.3U MDH\({}^{\star}\) under optimal reaction conditions (65 \({}^{\circ}\)C, 5.6% O\({}_{2}\) and 12.5% CO\({}_{2}\)). The reactions were initiated by the addition of NADH. All experiments were carried out in triplicate under the same conditions for different periods of time. An MDH\({}^{\star}\)-only reaction was run as a control (Figure 7B). The gluconic and malic acid yields from the cascade reaction were significantly higher than when MDH\({}^{\star}\) was used alone (Figure 7A). As a result, 15.96 mM malic acid and 9.94 mM gluconic acid were produced by the cascade reaction after 3 h compared to 0.309 mM malic acid from the MDH\({}^{\star}\)-only reaction. Notably, 2 mM malic acid was produced by the dual enzyme system after 15 min (which is higher than the malic acid yield by MDH\({}^{\star}\) alone after a reaction time of 3 h) and another high-value chemical, gluconic acid, was additionally produced by the cascade system from glucose and NAD\({}^{\star}\).