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 journalArticle

2 Citations (Scopus)

Abstract

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 K t 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 K si values for the oligogalacturonides were in the low mM range, and K i 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
Volume58
Issue number7
DOIs
StatePublished - Jul 1 2016

Fingerprint

Polygalacturonase
Acids
Gene encoding
Enzyme activity
Enzymes
Kinetic parameters
Escherichia coli
Genes
Amino Acid Sequence
Catalytic Domain
Amino Acids
Kinetics
Substrates
Processing
galacturonic acid
Amino acids
Proteins
pectin

Keywords

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

ASJC Scopus subject areas

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

Cite this

Wagschal, Kurt ; Rose Stoller, J. ; Chan, Victor J. ; Lee, Charles C. ; Grigorescu, Arabela A ; Jordan, Douglas B. / Expression and Characterization of Hyperthermostable Exo-polygalacturonase TtGH28 from Thermotoga thermophilus. In: Molecular Biotechnology. 2016 ; Vol. 58, No. 7. pp. 509-519.
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Expression and Characterization of Hyperthermostable Exo-polygalacturonase TtGH28 from Thermotoga thermophilus. / Wagschal, Kurt; Rose Stoller, J.; Chan, Victor J.; Lee, Charles C.; Grigorescu, Arabela A; Jordan, Douglas B.

In: Molecular Biotechnology, Vol. 58, No. 7, 01.07.2016, p. 509-519.

Research output: Contribution to journalArticle

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