## 4 Discussion  
To ensure a successful coupled reaction, the effects of GDH (the regeneration enzyme) reaction conditions on the activity of MDH\({}^{\star}\) (the production enzyme) were examined. The highest reduction in MDH\({}^{\star}\) enzymatic activity was caused by a decrease in HEPES buffer concentration from the standard 500 Mm HEPES in MDH\({}^{\star}\) assays to 100 Mm HEPES in GDH assays; the activity of MDH\({}^{\star}\) was reduced by 20% (Figure 2). The majority of enzymes require only moderate ionic strength, typically ranging between 50 and 200 Mm, but this is not the case for thermophilic enzymes which require buffer concentrations up to 1 M [15]. The observed decrease in activity is therefore likely due to the reduction in buffer capacity which, along with any added components, can reduce the obligatory enzyme activity [16]. Aside from HEPES, the addition of other GDH assay components such as MgCl\({}_{2}\), glycerol, and the GDH substrate, glucose, did not reduce MDH\({}^{\star}\) activity by more than 10% (Figure 2). MDH\({}^{\star}\) activity assays were carried out under 15% CO\({}_{2}\) and 5% O\({}_{2}\) atmospheric conditions; therefore, GDH activity was assayed under atmospheric conditions required by MDH\({}^{\star}\) and activity levels of GDH were measured via the concomitant production of NADH from the oxidation of glucose to gluconate. After 60 min, there was no significant difference between the amount of NADH produced by GDH assayed under aerobic conditions and GDH assayed under atmospheric control (Figure 3). From these findings, it was concluded that GDH is a suitable cofactor recycling partner and its reaction components do not drastically inhibit the activity of MDH\({}^{\star}\). It was concluded that GDH can be utilized effectively under altered atmospheric conditions as per the requirements of MDH\({}^{\star}\). For a fully feasible cofactor recycling coupling, the assay components of MDH\({}^{\star}\) were also tested on the activity of GDH under atmospheric conditions. This experiment demonstrated the drastic effect of pyruvate on reducing the activity of GDH (Figure 4). This loss of activity was determined to be due to the competitive inhibition of GDH by pyruvate, because concentrations of 10 mM pyruvate reduced GDH activity by \(\sim\)60%, and a twofold increase in pyruvate concentration increased the K\({}_{\text{M}}\) value sixfold (Supplementary Table S3). Concentrations of 30 mM pyruvate and 30 mM glucose were used to mitigate the issue of GDH inhibition by pyruvate. Cofactor recycling was demonstrated when using NAD\({}^{\star}\) or NADH as cofactors in the presence of both GDH and MDH\({}^{\star}\) (Table 3). In conclusion, we have demonstrated cofactor regeneration within a closed system of two thermophilic enzymes. The work carried out here sets the foundation for this concept to be explored further with longer reaction times and further optimization of reaction conditions. The redox neutrality of the two enzymes may now be exploited further by expanding the number of enzymes in the cascade system. Further optimization regarding the separation of the products can also be explored. Immobilization of the coupled enzymes will provide potential solutions to issues linked with enzyme cost and stability [17; 18]  
Figure 7: Substrate consumption and product formation by MDH\({}^{\star}\) and GDH (**A**) and MDH\({}^{\star}\) alone (**B**). and for catalyst recycling on an industrial scale [19], offering advantages of both economy and sustainability.