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Identification, Recombinant Expression, and Biochemical Analysis of Putative Secondary Product Glucosyltransferases from Citrus paradisi
Journal of Agricultural and Food Chemistry (2016)
  • Shivakumar P. Devaiah, East Tennessee State University
  • Daniel K. Owens, U.S. Department of Agriculture
  • Mebrahtu B. Sibhatu, East Tennessee State University
  • Tapasree Roy Sarkar, East Tennessee State Universtiy
  • Christy L. Strong, East Tennessee State University
  • Venkata K.P.S. Mallampalli, East Tennessee State University
  • Josephat Asiago, East Tennessee State University
  • Jennifer Cooke, East Tennessee State University
  • Starla Kiser, East Tennessee State University
  • Zhangfan Lin, East Tennessee State University
  • Anye Wamucho, East Tennessee State University
  • Deborah Hayford, East Tennessee State University
  • Bruce E. Williams, East Tennessee State University
  • Peri Loftis, East Tennessee State University
  • Mark Berhow, U.S. Department of Agriculture
  • Lee M. Pike, East Tennessee State Universtiy
  • Cecilia McIntosh, East Tennessee State University
Abstract
Flavonoid and limonoid glycosides influence taste properties as well as marketability of Citrus fruit and products, particularly grapefruit. In this work, nine grapefruit putative natural product glucosyltransferases (PGTs) were resolved by either using degenerate primers against the semiconserved PSPG box motif, SMART-RACE RT-PCR, and primer walking to full-length coding regions; screening a directionally cloned young grapefruit leaf EST library; designing primers against sequences from other Citrus species; or identifying PGTs from Citrus contigs in the harvEST database. The PGT proteins associated with the identified full-length coding regions were recombinantly expressed in Escherichia coli and/or Pichia pastoris and then tested for activity with a suite of substrates including flavonoid, simple phenolic, coumarin, and/or limonoid compounds. A number of these compounds were eliminated from the predicted and/or potential substrate pool for the identified PGTs. Enzyme activity was detected in some instances with quercetin and catechol glucosyltransferase activities having been identified.
Keywords
  • citrus paradisi,
  • flavonoid,
  • flavor,
  • glucosyltransferase,
  • heterologous expression
Publication Date
February 18, 2016
DOI
10.1021/acs.jafc.5b05430
Citation Information
Shivakumar P. Devaiah, Daniel K. Owens, Mebrahtu B. Sibhatu, Tapasree Roy Sarkar, et al.. "Identification, Recombinant Expression, and Biochemical Analysis of Putative Secondary Product Glucosyltransferases from Citrus paradisi" Journal of Agricultural and Food Chemistry Vol. 64 Iss. 9 (2016) p. 1957 - 1969 ISSN: 1520-5118
Available at: http://works.bepress.com/cecilia-mcintosh/23/