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

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

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. 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. 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.published_or_final_versio

Enzyme assays for lignin–carbohydrate bond hydrolysis

To identify, produce, and use enzymes, analytical methods known as enzyme assays are employed. Enzyme assays are based on analysing the changes brought about on a substrate by an enzyme under defined conditions. Assays are often based on simplified reactions acting as substitutes for the reaction of interest. In practice, this means that the ability to discover and use enzymes as biocatalysts is determined by the availability and applicability of such simplified reaction systems.The first part of biomass conversion is the degradation of lignified plant matter and the main bottleneck of this step is the non-destructive disassociation of polymeric biomass components. Some of the degradation recalcitrance is believed to be due to covalent bonds between the lignin and sugar components of the material (LC-bonds). Thus, enzymatic hydrolysis of these bonds can potentially improve component separation. So far, only a few enzymes capable of degrading LC-bonds have been identified. The low number may be due to the lack of enzyme assays for discovery and characterization.The purpose of this research effort has been to design assays for enzymes capable of breaking LC-bonds. The published works associated with this thesis describe various assay methods relevant to this goal: Paper I defines procedures for generating LC- bond-rich substrates from natural sources (lignin-carbohydrate complexes), with the aim of demonstrating their presence and detection by size-exclusion chromatography. Papers II and III describe synthetic-substrate assays for glucuronoyl esterases (GEs), the enzyme class with the best evidence of LC-bond hydrolysis. Paper II includes the synthesis of a β-diaryl ether for use as a GE assay substrate and Paper III presents and discusses several assays with different detection methods based on a commercially available GE substrate. Paper IV presents assays for enzyme synergy and shows how mass spectrometry can be used as an auxiliary detection method to better understand enzyme activities.This thesis places the enclosed articles into the overall context of LC-bond assays and describes possibilities for the combination of substrates, enzyme activities, and detection methods for the construction of novel LC-bond assays. As such, this work should offer background and a starting point for anyone wishing to do practical work on enzymatic LC-bond hydrolysis

Enzyme assays for lignin–carbohydrate bond hydrolysis

To identify, produce, and use enzymes, analytical methods known as enzyme assays are employed. Enzyme assays are based on analysing the changes brought about on a substrate by an enzyme under defined conditions. Assays are often based on simplified reactions acting as substitutes for the reaction of interest. In practice, this means that the ability to discover and use enzymes as biocatalysts is determined by the availability and applicability of such simplified reaction systems.The first part of biomass conversion is the degradation of lignified plant matter and the main bottleneck of this step is the non-destructive disassociation of polymeric biomass components. Some of the degradation recalcitrance is believed to be due to covalent bonds between the lignin and sugar components of the material (LC-bonds). Thus, enzymatic hydrolysis of these bonds can potentially improve component separation. So far, only a few enzymes capable of degrading LC-bonds have been identified. The low number may be due to the lack of enzyme assays for discovery and characterization.The purpose of this research effort has been to design assays for enzymes capable of breaking LC-bonds. The published works associated with this thesis describe various assay methods relevant to this goal: Paper I defines procedures for generating LC- bond-rich substrates from natural sources (lignin-carbohydrate complexes), with the aim of demonstrating their presence and detection by size-exclusion chromatography. Papers II and III describe synthetic-substrate assays for glucuronoyl esterases (GEs), the enzyme class with the best evidence of LC-bond hydrolysis. Paper II includes the synthesis of a β-diaryl ether for use as a GE assay substrate and Paper III presents and discusses several assays with different detection methods based on a commercially available GE substrate. Paper IV presents assays for enzyme synergy and shows how mass spectrometry can be used as an auxiliary detection method to better understand enzyme activities.This thesis places the enclosed articles into the overall context of LC-bond assays and describes possibilities for the combination of substrates, enzyme activities, and detection methods for the construction of novel LC-bond assays. As such, this work should offer background and a starting point for anyone wishing to do practical work on enzymatic LC-bond hydrolysis

Novel Enzymes for the degradation of Lignin-Carbohydrate complexes

In pulp and paper production, the delignification process damages the wood polymers irreversibly. A milder process based on enzymatic treatment could maintain various polymer properties that could be utilised for creating novel materials from the wood. One class of chemical bonds that needs to be hydrolysed in such a process are the hemicellulose-lignin bonds. These are thought to be of a heterologous mix of bonds consisting mostly of ether and ester bonds between hemicellulose sugars and the phenylpropane units of lignin (Jeffries 1990). Finding enzymes capable of hydrolysing these bonds are therefore of industrial relevance. In addition, hemicellulose-debranching enzymes that allows removal of lignin-substituted sidechains could be used to the same effect, why finding novel enzymes of this type is equally relevant. The present work outlines a process with which such novel enzymes for wood hydrolysis can be discovered by systematic screening of filamentous fungi under varying growth conditions

Novel Enzymes for the degradation of Lignin-Carbohydrate complexes

In pulp and paper production, the delignification process damages the wood polymers irreversibly. A milder process based on enzymatic treatment could maintain various polymer properties that could be utilised for creating novel materials from the wood. One class of chemical bonds that needs to be hydrolysed in such a process are the hemicellulose-lignin bonds. These are thought to be of a heterologous mix of bonds consisting mostly of ether and ester bonds between hemicellulose sugars and the phenylpropane units of lignin (Jeffries 1990). Finding enzymes capable of hydrolysing these bonds are therefore of industrial relevance. In addition, hemicellulose-debranching enzymes that allows removal of lignin-substituted sidechains could be used to the same effect, why finding novel enzymes of this type is equally relevant. The present work outlines a process with which such novel enzymes for wood hydrolysis can be discovered by systematic screening of filamentous fungi under varying growth conditions

Fungal Ferulic Acid Esterases – Specificity and Phylogeny

Ferulic Acid Esterases (FAE) is a large heterogeneous group of enzymes with activity on esters of hydroxy- and metoxy- substituted cinnamic acid derivatives, such as ferulic acid. These ester bonds occur in the cell walls of plants and are especially common in grasses. As little systematic knowledge has been collected about this group of enzymes and only a few enzymes have been biochemically characterised to date, we have explored the phylogeny of FAEs using bioinformatic tools. We can conclude that the known Ferulic Acid Esterases belong to several evolutionary distant groups, two of which have dozens of highly related sequences, and a few groups with no members other than the known enzyme. The phylogeny also suggests certain similarities of substrate specificity within groups and proposes enzymes, whose biochemical characterisation would be especially informative for our understanding of the FAE families