Part:BBa_K1448000

gamma-terpinene synthase

This part contains ɤ-terpinene synthase region of Citrus unshiu and was isolated by Dr.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 [http://ja.wikipedia.org/wiki/%E3%82%A6%E3%83%B3%E3%82%B7%E3%83%A5%E3%82%A6%E3%83%9F%E3%82%AB%E3%83%B3 Wikipedia, 2014,10,18]

Background and principles

Fig.1 Reaction catalyzed by ɤ-terpinene synthase. [http://iospress.metapress.com/content/ymr9r05l2k6j3ytb/ Suzuki et al, 2004]

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 ɤ-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 was cultivated and tried the detection by GC analysis.

Fig.3 GC-analysis data

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

Results

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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Growth curve

Fig.4 Transformant: BY4742 containing ɤ-terpinene generator inserted in p427-TEF, control: BY4742 containing empty p427-TEF

Introduction : Various mono-terpenoids are known to have an anti-microbial property. In order to evaluate the anti-microbial property of ɤ-terpinene, we made the growth curve of yeast cells containing ɤ-terpinene generator and compare to that of yeast cells without ɤ-terpinene generator.

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. OD600 was measured every 2 h.

Results : Yeast cells containing ɤ-terpinene generator showed slower growth than cells containing empty p427-TEF, indicating that ɤ-terpinene inhibits the growth of yeast cells.

Reference

• http://www.sciencedirect.com/science/article/pii/S0168945203003777 Shimada et al, 2003Takehiko Shimada, Tomoko Endo, Hiroshi, Fujii Masakazu Hara, Takanori Ueda, Masayuki Kita, Mitsuo Omura (2003) Molecular cloning and functional characterization of four monoterpene synthase genes from Citrus unshiu.
• http://pubs.acs.org/doi/abs/10.1021/jf020993f MARIO C. FOTI et al, 2003MARIO C. FOTI*,† AND K. U. INGOLD‡ (2003) Mechanism of Inhibition of Lipid Peroxidation by γ-Terpinene, an Unusual and Potentially Useful Hydrocarbon Antioxidant.

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]