Purification and Characterization of O2-insensitive Pyruvate Decarboxylase from Sulfolobus species

Abstract

Pyruvate decarboxylase (PDC) is a key enzyme in a two-step pathway for the production of ethanol. It catalyzes the non-oxidative decarboxylation of pyruvate to acetaldehyde in many mesophilic organisms. No conventional PDC has been found in hyperthermophiles, a group of microorganisms growing optimally at 80°C and above; however, bifunctional PDC/pyruvate ferredoxin oxidoreductase (POR) activities have been found to be present in several hyperthermophilic bacteria and archaea, but most of them are oxygen-sensitive and CoAdependent. It was reported that the CoA is not required for a recombinant PDC/POR from Sulfolobus tokodaii (Topt = 80ºC) and it is oxygen insensitive, but it’s not known why it has a much lower activity compared to other PDCs/PORs. Since PORs from hyperthermophilic crenarchaea Sulfolobus solfataricus (Topt = 80ºC) and Sulfolobus acidocaldarius (Topt = 80°C) are not oxygensensitive, it was hypothesized that they might be the most thermostable O2-insensitive PDCs. PDCs/PORs enzymes from S. solfataricus (Ss) and S. acidocaldarius (Sa) were purified using a fast performance liquid chromatography system (FPLC) anaerobically. POR activity was measured by monitoring the pyruvate-dependent reduction of benzyl viologen at 578 nm. PDC activity was measured by monitoring the pyruvate-dependent production of acetaldehyde. The acetaldehyde production was determined by using 2,4-dinitrophenylhydrazine (DNPH) derivatization method followed by high performance liquid chromatography (HPLC). Both enzymes from S. solfataricus and S. acidocaldarius were purified and SDS-PAGE showed that each heterodimeric enzyme had two subunits with a molecular mass of 37±3 kDa and 65±2 kDa respectively. S. solfataricus PDC and POR activities present in its cell-free extract (CFE) were determined to be 0.0027±0.0003 U/mg and 0.18±0.01 U/mg, respectively. Similarly, S. iv acidocaldarius PDC and POR activities present in its CFE were determined to be 0.0011±0.0004 U/mg and 0.10±0.01 U/mg, respectively. The enzyme from S. solfataricus was purified approximately 42-fold with a recovery of 25%, while the enzyme from S. acidocaldarius was purified approximately 70-fold with a recovery of 19%. Optimal pH for both S. solfataricus and S. acidocaldarius PORs were determined to be at pH 8.6, while optimal pH for their PDCs were 7.8. PDC/POR enzyme from S. solfataricus showed maximum activity at 80 ºC for PDC activity and at 90 ºC for POR activity; however, the optimum temperatures of PDC/POR from S. acidocaldarius were at 90 ºC for PDC activity and at 80 ºC for POR activity. PDCs/PORs enzymes from S. solfataricus and S. acidocaldarius were CoA dependent for both PDCs and PORs activities, a common feature of other PDCs/PORs except the one from S. tokodaii. Thermostability of the purified enzymes from S. solfataricus and S. acidocaldarius were determined by measuring the time required for losing 50% activity (t1/2) at 80 °C, which were approximately 2.9 h and 1.1 h respectively. It was determined that PDCs/PORs from S. solfataricus, and S. acidocaldarius were not oxygen sensitive. These thermostable and oxygen-stable PDCs may have great potential in applications in developing a more efficient system for bioethanol fermentation at high temperatures.