Hampus Sunner

Glucuronoyl Esterase Screening and Characterization Assays Utilizing Commercially Available Benzyl Glucuronic Acid Ester

Research on glucuronoyl esterases (GEs) has been hampered by the lack of enzyme assays based on easily obtainable substrates. While benzyl d-glucuronic acid ester (BnGlcA) is a commercially available substrate that can be used for GE assays, several considerations regarding substrate instability, limited solubility and low apparent affinities should be made. In this work we discuss the factors that are important when using BnGlcA for assaying GE activity and show how these can be applied when designing BnGlcA-based GE assays for different applications: a thin-layer chromatography assay for qualitative activity detection, a coupled-enzyme spectrophotometric assay that can be used for high-throughput screening or general activity determinations and a HPLC-based detection method allowing kinetic determinations. The three-level experimental procedure not merely facilitates routine, fast and simple biochemical characterizations but it can also give rise to the discovery of different GEs through an extensive screening of heterologous Genomic and Metagenomic expression libraries.

Synthesis and enzymatic hydrolysis of a diaryl benzyl ester model of a lignin-carbohydrate complex (LCC)

Abstract Specific degradation of the bonds between lignin and carbohydrates is an important step towards separating individual lignocellulosic biopolymers for sustainable production of materials and chemicals. One of the most established covalent lignin-carbohydrate (LC) interactions is the ester bond between the α- or γ-hydroxyl group of a lignin phenylpropane unit and a glucuronic acid side chain of xylan. In this work, a model of the LC benzyl ester bond was synthesized in a one-pot reaction from a β-O-4 lignin unit and d-glucuronic acid, both from commercial sources. The resulting lignin-carbohydrate complex (LCC) model was unstable in aqueous solution. However, at pH 4, the rate of spontaneous hydrolysis was sufficiently low to allow for enzymatic splitting experiments. The enzymatic hydrolysis of the LC benzyl ester bond of the LCC model was demonstrated by means of the glucuronoyl esterase StGE2 from Sporotrichum thermophile, which showed a preference for erythro forms of the LCC model.

Combining Substrate Specificity Analysis with Support Vector Classifiers Reveals Feruloyl Esterase as a Phylogenetically Informative Protein Group

Background Our understanding of how fungi evolved to develop a variety of ecological niches, is limited but of fundamental biological importance. Specifically, the evolution of enzymes affects how well species can adapt to new environmental conditions. Feruloyl esterases (FAEs) are enzymes able to hydrolyze the ester bonds linking ferulic acid to plant cell wall polysaccharides. The diversity of substrate specificities found in the FAE family shows that this family is old enough to have experienced the emergence and loss of many activities. Methodology/Principal Findings In this study we evaluate the relative activity of FAEs against a variety of model substrates as a novel predictive tool for Ascomycota taxonomic classification. Our approach consists of two analytical steps; (1) an initial unsupervised analysis to cluster the FAEs substrate specificity data which were generated by cultivation of 34 Ascomycota strains and then an analysis of the produced enzyme cocktail against 10 substituted cinnamate and phenylalkanoate methyl esters, (2) a second, supervised analysis for training a predictor built on these substrate activities. By applying both linear and non-linear models we were able to correctly predict the taxonomic Class (∼86% correct classification), Order (∼88% correct classification) and Family (∼88% correct classification) that the 34 Ascomycota belong to, using the activity profiles of the FAEs. Conclusion/Significance The good correlation with the FAEs substrate specificities that we have defined via our phylogenetic analysis not only suggests that FAEs are phylogenetically informative proteins but it is also a considerable step towards improved FAEs functional prediction.

Fungal glucuronoyl and feruloyl esterases for wood processing and phenolic acid ester/sugar ester synthesis

Feruloyl esterases (FAEs, E.C. 3.1.1.73, CAZy family CE1) and glucuronoyl esterases (GEs, E.C. 3.1.1.-, CAZy family CE15) are involved in the degradation of plant biomass by hydrolysing ester linkages in plant cell walls, and thus have potential use in biofuel production from lignocellulosic materials and in biorefinery applications with the aim of developing new wood-based compounds [1, 2]. GEs and FAEs are present in the genomes of a wide range of fungi and bacteria. Under conditions of low water content, these enzymes can also carry out (trans)esterification reactions, making them promising biocatalysts for the modification of compounds with applications in the food, cosmetic and pharmaceutical industry. Compared to the chemical process, enzymatic synthesis can be carried out under lower process temperatures (50-60°C) and results in fewer side products, thus reducing the environmental impact. We characterised new FAE and GE enzymes from mesophilic, thermophilic and coldtolerant filamentous fungi produced in Pichia pastoris. The enzymes were characterised for both their hydrolytic abilities on various model substrates (methyl ferulate, pNPferulate) - for potential applications in deconstruction of lignocellulosic materials and extraction of valuable compounds - as well as for their biosynthetic capacities. We tested and optimised the FAEs’ transesterification capabilities on ferulate esters in a 1- butanol-buffer system, with the aim of using the most promising candidates for the production of antioxidant compounds with improved hydrophobic or hydrophilic properties, such as prenyl ferulate, prenyl caffeate, glyceryl ferulate and 5-O-(transferuloyl)-arabinofuranose.