Purification and Characterization of O2-insensitive Pyruvate Decarboxylase from Sulfolobus species
dc.contributor.advisor | Ma, Kesen | |
dc.contributor.author | Alharbi, Faisal | |
dc.date.accessioned | 2020-06-17T19:56:43Z | |
dc.date.available | 2021-06-18T04:50:05Z | |
dc.date.issued | 2020-06-17 | |
dc.date.submitted | 2020-06-11 | |
dc.description.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 CoA-dependent. 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 oxygen-sensitive, 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 373 kDa and 652 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. 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. | en |
dc.identifier.uri | http://hdl.handle.net/10012/16000 | |
dc.language.iso | en | en |
dc.pending | false | |
dc.publisher | University of Waterloo | en |
dc.subject | pyruvate decarboxylase | en |
dc.subject | Ethanol fermentation | en |
dc.subject | pyruvate ferredoxin oxidoreductase | en |
dc.subject | Hyperthermophiles | en |
dc.subject | Sulfolobus | en |
dc.title | Purification and Characterization of O2-insensitive Pyruvate Decarboxylase from Sulfolobus species | en |
dc.type | Master Thesis | en |
uws-etd.degree | Master of Science | en |
uws-etd.degree.department | Biology | en |
uws-etd.degree.discipline | Biology | en |
uws-etd.degree.grantor | University of Waterloo | en |
uws-etd.embargo.terms | 1 year | en |
uws.contributor.advisor | Ma, Kesen | |
uws.contributor.affiliation1 | Faculty of Science | en |
uws.peerReviewStatus | Unreviewed | en |
uws.published.city | Waterloo | en |
uws.published.country | Canada | en |
uws.published.province | Ontario | en |
uws.scholarLevel | Graduate | en |
uws.typeOfResource | Text | en |