## 2 Results and Discussion
### Enzymatic Assay of His-Tag Purified VMPSTS and End Product Analysis  
Further clarification on the identities of the products produced was made by enzymatic assays analysis using His-tag purified VMPSTS proteins. Consistent with the previous crude VMPSTS result, germacrene D was still the major product comprising 48.9% of the total sesquiterpenes produced while \(a\)-copaene and \(\delta\)-cadinene contributed 9.5% and 8.8%, respectively (Figure 4). The mass spectra of the top three major products obtained based on peak area are shown in Figure 5. A host of other minor sesquiterpenes, undetected using the crude extract, was also produced albeit at lesser amounts when using the purified VMPSTS (Table 1). However, farnesene and aromadendrene which were detected in the crude protein extract analysis were not present in the purified protein analysis, suggesting that these could be intermediate metabolites or their derivatives resulting from the endogenous conversion of FPP to farnesol. Apart from the major peak at RT 19.44 min which was identified to be germacrene D, three other peaks at RT 18.19 min, 18.40 min and 18.66 min also had similar mass spectra and conferred germacrene D as the highest hit. This could represent four different stereoisomers of  
Figure 3: GC-MS chromatogram of products formed by crude VMPSTS protein at different retention times (RT). Negative control used consisted of NZ9000 host harboring empty pNZ8048 plasmid. At RT 6.00 min to 7.00 min: for VMPSTS, at 6.43: copaene, 6.62: farnesene, 6.71: aromadendrene, 6.77: 2-tridecanone, 6.84: germacrene D and 6.94: \(\delta\)- cadinene were observed while in the negative control at 6.62: nerylacetone, 6.67: 4,6-di-tert-butyl-m-cresol, 6.70: 1-dodecanol, 6.77: 2-tridecanone and 6.84: methyl 4,4,7-trimethyl-4,7-dihydroandan-6-carboxylate were noted. germacrene D which is not surprising considering germacrene D has 2 chiral centers in its carbon backbone. Furthermore, germacrenes have two endocyclic double bonds at 1(10)- and 4(5)- positions which could result in four geometric isomers [27]. It should also be noted that germacrene sesquiterpen are prone to Cope rearrangements induced by GC-MS conditions such as elevated temperature resulting in artefacts [27]. As such, the \(\beta\)-elemene peak at RT 17.56 min produced by the purified VMPSTS protein may not be a true product of VMPSTS. A few other sesquiterpenes were also produced but in negligible amounts (below 1%) and/or had match scores below 800 and thus were deemed ambiguous and are not reported here. Terpene synthases capable of producing multiple terpene products are very common due to rearrangements and quenching of the intermediate carbocations products [28,29]. The types of sesquiterpenes found in our research were in agreement with the reported _ab initio_ investigation of the cyclization of germacrene D which is usually accompanied by a host of cadinane and muurolane sesquiterpenoids [30]. All peaks were identified by comparison with the NIST and Wiley libraries only since standards for these specialized sesquiterpenes are not easily available. However, comparison of retention times with standards and isolation of the major compounds followed by NMR spectroscopy are suggested to be carried out as future work for structure determination which will strongly confirm the identities of the peaks identified by the NIST and Wiley libraries. Figure 4: GC-MS chromatogram showing sesquiterpenes produced from purified VMPSTS protein. The peaks in this chromatogram are listed in Table 1.