Expression and Characterization of Hyperthermostable Exo-polygalacturonase TtGH28 from Thermotoga thermophilus

Kurt Wagschal*, J. Rose Stoller, Victor J. Chan, Charles C. Lee, Arabela A. Grigorescu, Douglas B. Jordan

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

7 Scopus citations


D-galacturonic acid is a potential platform chemical comprising the principal component of pectin in the citrus processing waste stream. Several enzyme activities are required for the enzymatic production of galacturonic acid from pectin, including exo- and endo-polygalacturonases. The gene TtGH28 encoding a putative GH28 polygalacturonase from Pseudothermotoga thermarum DSM 5069 (Theth_0397, NCBI# AEH50492.1) was synthesized, expressed in Escherichia coli, and characterized. Alignment of the amino acid sequence of gene product TtGH28 with other GH28 proteins whose structures and details of their catalytic mechanism have been elucidated shows that three catalytic Asp residues and several other key active site residues are strictly conserved. Purified TtGH28 was dimeric and hyperthermostable, with Kt 0.5 = 86.3 °C. Kinetic parameters for activity on digalacturonic acid, trigalacturonic acid, and polygalacturonic acid were obtained. No substrate inhibition was observed for polygalacturonate, while the Ksi values for the oligogalacturonides were in the low mM range, and Ki for product galacturonic acid was in the low μM range. Kinetic modeling of the progress of reaction showed that the enzyme is both fully exo- and fully non-processional.

Original languageEnglish (US)
Pages (from-to)509-519
Number of pages11
JournalMolecular Biotechnology
Issue number7
StatePublished - Jul 1 2016


  • GH28
  • Galacturonic acid
  • Hyperthermostable
  • Pectin
  • Polygalacturonase
  • Product inhibition
  • Substrate inhibition

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biochemistry
  • Applied Microbiology and Biotechnology
  • Molecular Biology


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