Partial Purification and Characterization of a Monoterpene Synthase Extracted from Young Leaves of Michellia Alba
Lee, Yuan Chern (2006) Partial Purification and Characterization of a Monoterpene Synthase Extracted from Young Leaves of Michellia Alba. Masters thesis, Universiti Putra Malaysia.
This study was conducted to partially purify linalool synthase from the young leaves of Michellia alba (Cempaka Putih) from the Magnoliaceae family. The technique used to determine the amount of linalool produced from enzyme activity was the combination of solid-phase microextraction (SPME) and gas chromatography with flame-ionization detecter (GCFID) technique. The substrate used for this enzyme activity assay was geranylpyrophosphate (GPP). Optimal conditions such as temperature and incubation time for SPME technique were also determined. The linalool synthase exhibited a strict requirement for a divalent metal cofactor with a preference for Mg2+, Mn2+ and K+ ions. The optimal pH and temperature of the enzyme was 6.0 and 30oC respectively. The enzyme was inhibited by 1,2-Di(2-aminoethoxy)ethane-N,N,N’N’-tetra-acetic acids (EGTA). Three steps of partial purification of enzyme were carried out, including the crude extraction of young leaves, ultra centrifugation and Mono-Q anion exchange chromatography. The partially purified linalool synthase was characterized and studied for its enzyme kinetic properties. The linalool synthase has a Km of 83µM for substrate GPP. The SPME-GCFID technique was later proved to be reliable and sensitive in determination of monoterpene products.
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