Enzymatic fingerprinting and modification of acetylated pectins

C.A. Remoroza

Research output: Thesisinternal PhD, WU

Abstract

To reveal the ester distribution patterns in acetylated pectins, an enzymatic fingerprinting method usinga combined endo-polygalacturonase (PG) and pectin lyase (PL) treatment followed by hydrophilicinteraction liquid chromatography coupled to electrospray ionization ion trap mass spectrometry with evaporative light scattering detection was developed. This methodpaved the way for the development of the new quantitative parameters degree of hydrolysis by PG (DHPG) and degree of hydrolysis by PL (DHPL). These parameters distinguished the methylester and acetyl group distribution patterns within different sugar beet pectins (SBPs). In the case of pectin having a degree of methylesterification (DM) of >50 and acetylation of ~20, the above approach was insufficient. Hence, a seconddigestion was introduced using  a fungal pectin methylesterase and a PG.More than 60% of the total GalA residues present in three SBPs were recovered as monomer and oligomers after the two digestions. The first digestion of the acid extracted commercial SBP revealed the presence of small blocks of nonesterified GalA residues and segments containing large blocks of PL degradable methylesterified and /or acetylated GalA residues. Blocks of partly methylesterified, non-acetylated GalA residues were recognized after the second digestion. These results show that the acetylation pattern is non-random.

A pectin acetylesterase (BliPAE) and a pectin methylesterase (BliPME) from Bacillus licheniformis DSM13 were produced, purified and biochemically characterized. The mode of action of BliPAE and BliPME towards acetylated pectins was revealed using the newly developed enzymatic fingerprinting method. BliPAE specifically deacetylates the O-3 linked acetyl groups of nonmethylesterified galacturonic acid residues in the homogalacturan of pectin. BliPME efficiently de-methylesterifies lemon pectins (DM34-76 ®DM 0) and SBPs (DM 30-73 ®DM 14) in a blockwise manner. BliPME is quite tolerant towards the acetyl groups present within the SBPs. For the first time, a comprehensive experimental characterization was directed to enzymes from B. licheniformis having a PAE and a PME activity.

Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
Supervisors/Advisors
  • Gruppen, Harry, Promotor
  • Schols, Henk, Promotor
Award date21 Mar 2014
Place of PublicationWageningen
Publisher
Print ISBNs9789461739025
DOIs
Publication statusPublished - 21 Mar 2014

Keywords

  • pectins
  • acetylation
  • pectin lyase
  • polygalacturonase
  • characterization
  • methodology

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