Mushroom (Agaricus bisporus) polyphenoloxidase (PPO) (EC 1.14.18.1) was
isolated and purified using an aqueous two phase system composed of octyl phenol
(ethyleneglycol) 7-8 ether (Triton X-114/TX-114). TX-114 is a non-ionic surfactant
which thermoseparates in water and forms an aqueous two phase system with a
surfactant-depleted top phase and a surfactant-enriched bottom phase. Critical micelle concentration (CMC) and cloud point of the surfactant are 0.17 mM and
22º / C respectively. The partitioning behavior of mushroom PPO in water/TX-114
aqueous two-phase systems was studied and the effects of TX-114 concentration,
ionic strength, pH, temperature and crude extract preparation on PPO partitioning
were studied. PPO generally partitioned to the surfactant-depleted top phase / whereas
many other hydrophobic proteins and molecules partitioned to the surfactantenriched
bottom phase. When two-step ultrafiltration was used as a pretreatment,
complete enzyme recovery was achieved with all studied TX-114 concentrations.
Moreover, about 5 fold purification was achieved by using 8% TX-114. The
purification increased to 10 fold by using polyvinylpolypyrolidone (PVPP) at pH 7.0
with a recovery of 72%. However changing pH from 7.0 to 6.0 increased the
purification factor and enzyme recovery to 15 fold and 100%, respectively. Addition
of potassium or sodium salts caused PPO molecules to partition in the surfactantenriched
bottom phase. Finally, crude enzyme can be concentrated besides being
purified and recovered by doing aqueous two-phase separation at room temperature.
| Identifer | oai:union.ndltd.org:METU/oai:etd.lib.metu.edu.tr:http://etd.lib.metu.edu.tr/upload/1262971/index.pdf |
| Date | 01 January 2003 |
| Creators | Besel, Elif |
| Contributors | Bakir, Ufuk |
| Publisher | METU |
| Source Sets | Middle East Technical Univ. |
| Language | English |
| Detected Language | English |
| Type | M.S. Thesis |
| Format | text/pdf |
| Rights | To liberate the content for public access |
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