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Study on in vitro NR biosynthesis by rapid quantitative determination of substrate depletion

Yuwen Guo,Kexu Bao,Dongli Han,Xiaohui Wu,Shuangbing Li,Yiyang Dong,Jichuan Zhang

A convenient and nonradioactive method for quantifying in vitro NR biosynthesis is presented that is based upon the quantitation of substrate depletion by ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). NR oligomers could be in vitro biosynthesized with the enzyme source from Hevea brasiliensis ( Hevea ) or Taraxacum kok-saghyz ( TKS ) by exogenous monomers (IPP) and initiators (FPP). The IPP incorporation rate and FPP consumption rate were 62.24% and 51.14% respectively when the washed rubber particles (WRP) of Hevea was the enzyme source. The IPP incorporation rate and the FPP consumption rate were 74.49% and 95.90% respectively when the sediment bottom fraction (BF) of Hevea was the enzyme source. The in vitro NR biosynthesis can be divided into two stages:(1) the initiation reaction of FPP, which occurs more in BF, and (2) the growth reaction of IPP, which occurs more in WRP. In addition, the IPP incorporation and FPP consumption rates were 59.39% and 34.15% respectively when the BF of TKS was selected as an enzyme source.