Enzyme assays for lignin–carbohydrate bond hydrolysis

Doctoral thesis, 2016

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.