Part:BBa_K1448000
gamma-terpinene synthase
This part contains ɤ-terpinene synthase region of Citrus unshiu and was isolated by Mr.Shimada from Citrus unshiu.http://www.sciencedirect.com/science/article/pii/S0168945203003777 Shimada et al, 2003
Further information:
- DDBJ entry: http://getentry.ddbj.nig.ac.jp/getentry/na/AB110639/?format=flatfile&filetype=html&trace=true&show_suppressed=false&limit=10 AB110639
- Origin of the enzyme: Citrus unshiu
Background and principles
Characterization
Western-bloting analysis
Yeast cells carrying plasmids of p427-TEF-GST-monoterpenoid synthase were cultivated overnight in SD medium (0.67% yeast nitrogen base and 2% glucose with G418) 50mL at 28°C. Cells were collected and kept at -30˚C. Western blot analysis was carried out using anti-GST antibody and 12.5% polyacrylamide gel with SDS.
<Results>
Western blot data (Fig.2) showed clear bands around 90kDa in the lanes of p427-TEF- monoterpenoid synthase but not in the lane of empty vector.
In vivo detection of ɤ-terpinene using GC
Introduction
Since -terpinene is a volatile material, we expected that an amount of ɤ-terpinene were detected at the headspace of the culture in which the transformant with ɤ-terpinene generator is cultivated and tried the detection by GC analysis.
GC analysis ofɤ-terpinene in the culture
Materials and Methods
We cultivated the transformants containing ɤ-terpinene generator in p427-TEF or only empty p427-TEF in 50 mL SD medium (0.67% yeast nitrogen base and 2% glucose with G418) at 29˚C, 120 rpm for 24 h.
Analytic instrument: Shimadzu GC14A
Column: GLScience InertCap1 (15m Length,0.25 mm I.D., 0.25mm film thickness)
Injection port 200C, Detector port 210 C
Detector: Flame Ionization Detector
Column Oven Temperature 40C/5min- 10C/min-200C/5min
Carrier Gas: Helium
Result
we uesd SPME (SOLID PHASE MICROEXTRACTION) to catch ɤ-terpinene at the headspace of the culture. However, any amounts of ɤ-terpinene were not detected at the headspace of the culture (the exposure time is 30min).
Next we tried to catch ɤ-terpinene in the liquid phase of the culture using monotrap (the exposure time is 1h) and an amount of ɤ-terpinene was detected in the liquid phase of the culture.
Reference
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 204
Illegal XhoI site found at 715 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
n/a | gamma-terpinene synthase |