Characterisation and mode of action of an exopolygalacturonase from the hyperthermophilic bacterium Thermotoga maritime

L.D. Kluskens, G.J.W.M. van Alebeek, J. Walther, A.G.J. Voragen, W.M. de Vos, J. van der Oost

Research output: Contribution to journalArticleAcademicpeer-review

36 Citations (Scopus)

Abstract

An intracellular pectinolytic enzyme, PelB (TM0437), from the hyperthermophilic bacterium Thermotoga maritima was functionally produced in Escherichia coli and purified to homogeneity. PelB belongs to family 28 of the glycoside hydrolases, consisting of pectin-hydrolysing enzymes. As one of the few bacterial exopolygalacturonases, it is able to remove monogalacturonate units from the nonreducing end of polygalacturonate. Detailed characterization of the enzyme showed that PelB is highly thermo-active and thermostable, with a melting temperature of 105 °C and a temperature optimum of 80 °C, the highest described to date for hydrolytic pectinases. PelB showed increasing activity on oligosaccharides with an increasing degree of polymerization. The highest activity was found on the pentamer (1000 U·mg1). In addition, the affinity increased in conjunction with the length of the oligoGalpA chain. PelB displayed specificity for saturated oligoGalpA and was unable to degrade unsaturated or methyl-esterified oligoGalpA. Analogous to the exopolygalacturonase from Aspergillus tubingensis, it showed low activity with xylogalacturonan. Calculations on the subsite affinity revealed the presence of four subsites and a high affinity for GalpA at subsite +1, which is typical of exo-active enzymes. The physiological role of PelB and the previously characterized exopectate lyase PelA is discussed.
Original languageEnglish
Pages (from-to)5464-5473
JournalFEBS Journal
Volume272
Issue number21
DOIs
Publication statusPublished - 2005

Keywords

  • site-directed mutagenesis
  • aspergillus-niger
  • crystal-structure
  • butyrivibrio-fibrisolvens
  • endopolygalacturonase ii
  • subsite affinities
  • genome sequence
  • bovine rumen
  • sp-nov
  • pectin

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