HPLC determination of activity of UGT75B1、UGT71B6、UGT71C5 and the kinetic analysis
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    Abstract:

    The cDNA sequences of UGT75B1, UGT71B6, UGT71C5 were cloned by PCR method and then were constructed into glutathione S-transferase gene fusion vector PGEX-6P-1. All of the plasmids were transformed into E. coli strain BL21 individually for recombinant protein expression. Recombinant UGTs were purified from supernatant of cell lysates. To investigate the activity of recombinant UGTs, HPLC was employed to determine their activity to catalyze ABA and p-aminobenzoic acid. Analysis by reverse-phase HPLC was carried out using a Kromasil C18 column (250mm×4.6mm, 5μm) and glucose ester was separated by a linear gradient of 10%-100% methanol in H2O (all solutions contained 2.5ml/L of acetic acid and 0.4ml/L of triethylamine) or 10%-20% acetonitrile in H2O (all solutions contained 1ml/L of trifluoroacetic acid). The results showed that only UGT75B1 presented strong p-aminobenzoic acid acylglucosyltransferase activity and the others had lower activity. All of the recombinant UGTs had ABA-GE activity. However, UGT71B6 performed highest activity. Analysis of Michaelis-Menten kinetics of UGT75B1, UGT71B6 and UGT71C5 indicated that their Km was 0.73, 0.43 and 0.45 mM, respectively. In conclusion, our investigations demonstrate that UGT75B1 has higher p-aminobenzoic acid acylglucosyltransferase activity than ABA-GE activity. It seems that p-aminobenzoic acid is the specific substrate of UGT75B1. In comparison, UGT71B6 and UGT71C5 with similar Km perform higher ABA-GE activity.

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Cite this article as: CEN Yong-Yi, LIU Zheng, LI Xu-Feng. HPLC determination of activity of UGT75B1、UGT71B6、UGT71C5 and the kinetic analysis [J]. J Sichuan Univ: Nat Sci Ed, 2016, 53: 169.

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History
  • Received:April 21,2014
  • Revised:May 12,2014
  • Adopted:May 13,2014
  • Online: May 30,2016
  • Published: