Engineering & Discovery of CAZyme AA5_2 Oxidases

Filip Mollerup

Research output: ThesisDoctoral ThesisCollection of Articles


The carbohydrate active enzyme (CAZyme) database includes carbohydrate oxidases that have been classified into various auxiliary activity (AA) families. Among these, copper-radical oxidases that target primary hydroxyls are grouped into the AA5_2 subfamily. The most well-characterized to date is galactose oxidase from the aggressive plant pathogenic fungus Fusarium graminearum (FgrGaOx). FgrGaOx is one of the most utilized biocatalysts for detection or oxidation of primary hydroxyls in galactose-containing carbohydrates and aliphatic alcohols due to its robustness, high turnover, and use of atmospheric di-oxygen as the terminal electron acceptor, allowing generation of aldehyde products in pH-neutral aqueous solutions at room temperature. The enzyme has been studied and applied in a wide range of green processes, most notably for specific activation of oligo- and polysaccharides in carbohydrate engineering for hydro- and aerogels, carbohydrate-based films for food packaging, and coating of paper surfaces with oxidized hemicellulose. However, applications of FgrGaOx are limited to galactose-containing carbohydrates as the enzyme strictly oxidizes galactosyl residues. This thesis aims to discover and develop family AA5_2 carbohydrate oxidases with distinct activity relative to FgrGaOx. First, an engineering approach was applied to enhance FgrGaOx action. This approach included site-directed mutagenesis and construction of fusion proteins comprising galactomannan or cellulose binding domains. Impacts of fusing the carbohydrate binding modules (CBMs) to FgrGaOx were consistently higher when fusing the CBM to the C-terminus of the enzyme. While CBM fusion increased substrate binding and confirmed that FgrGaOx remained active following adsorption to cellulosic materials, it did not increase the end-point oxidation of polysaccharides by FgrGaOx. Moreover, site directed mutagenesis of FgrGaOx-CBM fusions revealed an apparent trade-off between the catalytic efficiency and substrate range of the enzyme. Comprehensive consideration of characterized AA5_2 oxidases, including engineered variants of FgrGaOx, identified amino acid positions important to catalysis, substrate selectivity, and catalytic efficiency. Resulting sequence-function relationships were used to identify consensus patterns among copper-radical oxidases in AA5_2. This analysis informed the selection of two new AA5_2 members, namely CgrRaOx from Colletotrichum graminicola and PruAA5_2A from Penicillium rubens Wisconsin 54-1255, for detailed biochemical characterization. In both cases, the substrate preference of the enzymes was clearly distinguished from that of known galactose oxidases. In particular, despite sharing only 23% identity, CgrRaOx and PruAA5_2A preferred raffinose and glycolaldehyde dimer substrates. This discovery uncovered a new connection across the AA5 family, and sheds new light on the biological roles of AA5_2 oxidases.
Translated title of the contributionEngineering & Discovery of CAZyme AA5_2 Oxidases
Original languageEnglish
QualificationDoctor's degree
Awarding Institution
  • Aalto University
  • Master, Emma, Supervising Professor
Print ISBNs978-952-60-8309-4
Electronic ISBNs978-952-60-8310-0
Publication statusPublished - 2018
MoE publication typeG5 Doctoral dissertation (article)


  • galactose oxidase
  • protein engineering
  • CAZYme
  • enzyme discovery


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