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

Image of Citrus unshiu

Background and principles

Fig.2 Reaction catalyzed by ɤ-terpinene synthase.

Characterization

Western-bloting analysis

(Fig.2 Western-bloting analysis of GST and d-limonene synthase (Lane2), ɤ-terpinene synthase (Lane3) and β-pinene synthase(Lane4) fusion proteins expressed in yeast. (A) The gel picture of SDS-PAGE and western-blot; Lanes: 1, p427-TEF; 2, p427-TEF-LS; 3, p427-TEF-ɤTPNS; 4, p427-TEF-βPINS. (B) vector map of plasmids. (C) The names and molecular weights of each mono-terpene synthase.)

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.

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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 ofɤ-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.

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Reference

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 204
  Illegal XhoI site found at 715
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]