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Biotechnology Letters

, Volume 39, Issue 7, pp 1009–1018 | Cite as

Molecular and biochemical characterization of squalene synthase from Siraitia grosvenorii

  • Heling Su
  • Yongming Liu
  • Yalun Xiao
  • Yanlian Tan
  • Yunyan Gu
  • Bin Liang
  • Hongli Huang
  • Yaosheng Wu
Original Research Paper

Abstract

Objectives

To clone and characterize the squalene synthase from Siraitia grosvenorii (SgSQS).

Results

The gene encoding SgSQS was cloned. SgSQS has 417 amino acid residues with an pI of 7.3. There are 32 phosphorylation sites in its sequence: S48 as well as S196 play important roles in regulation of enzyme activity. The enzyme is a monomeric protein with a cave-like active center formed by α helixes and has two transmembrane domains at its C-terminus. SgSQS mRNA expression in stem and root were about twice as much as that in leaf and peel. Full-length SgSQS with measurable catalytic activity was expressed in Escherichia coli. SgSQS activity was optimal at 37 °C and pH 7.5 respectively.

Conclusion

SgSQS gene was cloned, and the molecular structure and biochemical function of SgSQS were characterized.

Keywords

Cloning Enzymatic properties Gene expression Sequence analysis Siraitia grosvenorii Squalene synthase 

Notes

Acknowledgements

This work was supported by the National Natural Science Foundation of China (31260069) and the Training Project of the Outstanding Higher Education Teachers of Guangxi (2014) supported by the Education Department of Guanxi Zhuang Autonomous Region, China. We thank Dr. Shanhong Ling at Monash University, Melbourne, Australia, for his help in the manuscript preparation and edition.

Supporting information

Supplementary Fig. 1—Phylogenetic tree of the squalene synthases from Siraitia grosvenorii and other terrestrial plants.

Supplementary Fig. 2—Analysis of the tissue-specific expression of the squalene synthase from Siraitia grosvenorii using qRT-PCR.

Supplementary Fig. 3—Activity assay of the recombinant squalene synthase from Siraitia grosvenorii (SgSQS) for determination of K m and V max values.

Supplementary material

10529_2017_2328_MOESM1_ESM.docx (395 kb)
Supplementary material 1 (DOCX 394 kb) Supplementary Fig. 1 Phylogenetic tree of the squalene synthases from Siraitia grosvenorii and other terrestrial plants. The tree was constructed by the neighbor-joining method using MEGA 5.0 program based on the deduced amino acid sequences of these plants. Bootstrap values of >70% are indicated at each node, and GenBank accession numbers and clone numbers are showed on the branches of the tree
10529_2017_2328_MOESM2_ESM.docx (35 kb)
Supplementary material 2 (DOCX 35 kb) Supplementary Fig. 2 Analysis of the tissue-specific expression of the squalene synthase from Siraitia grosvenorii using qRT-PCR
10529_2017_2328_MOESM3_ESM.docx (153 kb)
Supplementary material 3 (DOCX 153 kb) Supplementary Fig. 3 Activity assay of the recombinant squalene synthase from Siraitia grosvenorii (SgSQS) for determination of K m and V max values. a Michaelis–Menten plot of SgSQS activity on FPP. b Lineweaver–Burk plot of SgSQS activity on FPP. c Michaelis–Menten plot of SgSQS activity on NADPH. d Lineweaver–Burk plot of SgSQS activity on NADPH

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Copyright information

© Springer Science+Business Media Dordrecht 2017

Authors and Affiliations

  1. 1.Department of Biochemistry and Molecular BiologyGuangxi Medical UniversityNanningChina
  2. 2.Key Laboratory of Medicinal Biotechnology, Guilin Medical UniversityGuilinChina

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