Yeast cell-free enzyme system for bio-ethanol production at elevated temperatures

A yeast cell-free enzyme system containing an intact fermentation assembly and that is capable of bio-ethanol production at elevated temperatures in the absence of living cells was developed to address the limitations associated with conventional fermentation processes. The presence of both yeast glycolytic and fermentation enzymes in the system was verified by SDS-PAGE and LC-MS/MS Q-TOF analyses. Quantitative measurements verified sufficient quantities of the co-factors ATP (1.8 mM) and NAD+ (0.11 mM) to initiate the fermentation process. Bio-ethanol was produced at a broad temperature range of 30-60 °C but was highly specific to a pH range of 6.0-7.0. The final bio-ethanol production at 30, 40, 50, and 60 °C was 3.37, 3.83, 1.94, and 1.60 g/L, respectively, when a 1% glucose solution was used, and the yield increased significantly with increasing cell-free enzyme concentrations. A comparative study revealed better results for the conventional fermentation system (4.46 g/L) at 30 °C than the cell-free system (3.37 g/L); however, the efficacy of the cell-free system increased with temperature, reaching a maximum (3.83 g/L) at 40 °C, at which the conventional system could only produce 0.48 g/L bio-ethanol. Successful bio-ethanol production using a single yeast cell-based enzyme system at higher temperatures will lead to the development of novel strategies for efficient bio-ethanol production through SSF.