Lytic polysaccharide monooxygenases from Myceliophthora thermophila C1

Matthias Frommhagen

Research output: Thesisinternal PhD, WU

Abstract

Current developments aim at the effective enzymatic degradation of plant biomass polysaccharides into fermentable monosaccharides for biofuels and biochemicals. Recently discovered lytic polysaccharide monooxgygenases (LPMOs) boost the hydrolytic breakdown of lignocellulosic biomass, especially cellulose, due to their oxidative mechanism. At the beginning of this thesis, only few LPMOs were characterized and many aspects related to their catalytic performance were unknown. Hence, in this thesis, we investigated AA9 LPMOs from Myceliophthora thermophila C1. This fungus encodes 22 putative AA9 LPMOs and we hypothesized that these enzymes differ in their substrate preference and mode of action towards plant cell wall polysaccharides.

We demonstrated that MtLPMO9A oxidizes xylan associated to cellulose, which is the only published LPMO comprising this capability known so far. We also showed that MtLPMOs from M. thermophila C1 differ in their substrate preference and C1-/C4-regioselectivity. Moreover, we described the use of reversed phase (RP)-UHPLC in combination with non-reductive 2-aminobenzamide (2-AB) labeling to separate and identify C4-oxidized gluco-oligosaccharides.

All characterized MtLPMOs differ in their reducing agent preference. The highest amount of non-oxidized and oxidized gluco-oligosaccharides from cellulose were released by MtLPMOs in the presence of reducing agents with a 1,2-benzenediol or 1,2,3-benzenetriol moiety. The latter compounds can be formed from lignin-building blocks by using polyphenol oxidases (PPOs), such as MtPPO7 from M. thermophila C1, which boost the LPMO-driven lignocellulose oxidation. Sequence analysis of genomes of 336 Ascomycota and 208 Basidiomycota revealed a high correlation between MtPPO7-like and AA9 LPMO-like genes. Finally, a β-glucosidase-assisted method was developed to quantify the catalytic performance of the C1-oxidizing MtLPMO9B and MtLPMO9D. The catalytic performance of both MtLPMOs was strongly dependent on pH and temperature. Notably, pH mainly affected the reducing agent dependency whereas temperature influenced the operational stability of both MtLPMOs.

In summary, our study contributed to the further understanding of LPMO-driven lignocellulose degradation.

Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
Supervisors/Advisors
  • Gruppen, H., Promotor
  • van Berkel, Willem, Promotor
  • Kabel, Mirjam, Co-promotor
Award date8 Sep 2017
Place of PublicationWageningen
Publisher
Print ISBNs9789463436434
DOIs
Publication statusPublished - 2017

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