## 2 Results and Discussion
### Enzymatic Assay of His-Tag Purified VMPSTS and End Product Analysis
\begin{table}
\begin{tabular}{l c c c} \hline
**Sesquiterpenes** & **Retention Time** & **Match score** & **Percentage** \\ \hline \(\delta\)-Elemene & 16.55 & 950 & 2.4 \\ Ylangene & 17.26 & 955 & 4.5 \\ \(\alpha\)-Copaene & 17.40 & 943 & 9.5 \\ \(\delta\)-Elemene & 17.56 & 922 & 2.2 \\ Germacrene D & 18.19 & 903 & 5.3 \\ Germacrene D & 18.40 & 913 & 3.7 \\ Germacrene D & 18.66 & 890 & 2.1 \\ \(\alpha\)-Gurjunene & 18.74 & 905 & 4.2 \\ \(\varepsilon\)-Muurolene & 18.80 & 824 & 1.2 \\ \(\alpha\)-Caryophyllene & 18.89 & 946 & 2.1 \\ Germacrene D & 19.44 & 966 & 48.9 \\ \hline \end{tabular}
\end{table} 
Table 1: Summary of retention times, match score and percentages of sesquiterpenes produced. Figure 5: Mass spectra of top three sesquiterpenes produced by recombinant VMPSTS in comparison with the corresponding mass spectra from the NIST or Wiley Library. Top panel shows mass spectra of the sample compound while bottom panel shows mass spectra of the compound in the library. (**a**) is germacrene D; **(b)** is \(\alpha\)-copaene; and (**c**) is \(\delta\)-cadinene. Previously, it was reported that nerolidol was the only sesquiterpene detected at 0.5% of the total scent, when analyzed directly from open VMP flowers using SPME sampling [22]. Copaene and germacrene D were also found in the essential oils of VMP at 0.17% and 0.13% respectively when the compounds from open VMP flowers soaked in hexane overnight were analyzed using GC-MS [31]. The terpenoid composition obtained directly from the open flowers of VMP differs slightly from the products produced by the _in vitro_ recombinant VMPSTS study. One salient observation was that cadinene, ylangene, muurolene and muurolol, caryophyllene, elemene, and gurjunene found in the _in vitro_ study were not detected either in the scent or the essential oils of VMP open flowers. This could be due to the low amounts produced in the plants or the sampling method used which could subject the major sesquiterpenes to chemically rearrange in different ways than in the _in vitro_ studies, resulting in different compositions of minor sesquiterpene compounds. In addition, types of hosts may also influence composition of products [32]. There are also many other terpen found in VMP open flowers especially monoterenes which contribute to the fragrance of the plant. However, the terpene synthases responsible for these terpenoids have not been isolated and characterized yet. When the VMPSTS protein sequence was subjected to a BLAST search, \(\alpha\)-humulene (synonym \(\alpha\)-caryophyllene), germacrene-D and \(\delta\)-cadinene terpenhes were among the top hits with up to 48% identity, along with other unidentified sesquiterpene synthases. This shows that the protein sequence of VMPSTS is closely related to similarly functional sesquiterpene synthases from other plants such as ginger (_Zinger zerumbet_ and _Zinger officinal_) and castor oil plant (_Ricinus communis_). Although germacrene D synthases has been isolated and characterized from other plants such as ginger (_Zingiber officinal_) [29] and grapevine (_Vitis vinefera_) [33], both enzymes and the one used in this study displayed different product compositions. The germacrene D synthase isolated from _Zingiber officinal_ produced germacrene D and germacrene B as its major product along with nine co-products including germacrene C, \(\alpha\)-humulene and _trans_-nerolidol. The germacrene D synthase from _Vitis vinefera_ on the other hand only produced germacrene D as a major product and \(\delta\)-cadinene as its minor product. The differences in the product compositions of germacrene D synthases from different plants demonstrate the vast array of isoprenoid variation and the versatility of the terpene synthases producing them. In terms of bioactivity, germacrene D has been reported to have insectical effects against mosquitoes [34] and to repel aphids [35]. However, it is an attractant to the Tobacco Mudworm moth, _Heliothis virescens_[36].