| 内容提要: |
We developed a
colorimetric assay using
yeast inorganic pyrophosphatase (IPP1) as a coupling enzyme to measure the activities of
terpene synthases. IPP1 hydrolyzes pyrophosphate, the byproduct
of terpene synthase
catalyzed reactions, into orthophosphate,
which can then be quantitated by reacting with molybdic acid to form a blue
color compound. As a proof of concept, this method was used to quantitatively
characterize three santalene synthases,
SaSSy and SspiSSy involved in sandalwood oil biosynthesis, and a phylogenetically distant SanSyn from Clausena lansium. Our study provided the
kinetic parameters of all three santalene synthases and demonstrated the
validity of the enzyme couple colorimetric assay by the comparison of this
assay with the existing GC-MS method. |