Characterisation of the enzymatic degradation of lignin
Item statusRestricted Access
Embargo end date10/06/2023
Lignin is one of the most abundant and diverse biopolymers on the planet, comprising approximately one third of all woody plant matter. This complex, non-repeating, heteroaromatic polymeric structure provides endless potential as an alternative, renewable feedstock to petrochemicals to produce fine chemicals. Lignin has been previously utilised to produce high value chemicals such as vanillin, however it is primarily considered a waste product and burned to fuel paper mill processes. As the world moves towards a circular economy, this abundant resource can no longer be neglected as a waste product and should be utilised as a replacement for fossil fuels in the synthesis of fine chemicals. In this regard, the use of ligninolytic enzymes has long been touted as the primary mechanism by which to achieve lignin valorisation because they act as more selective green catalysts that can perform an array of functions that are not always replicable by conventional chemistry. In Chapter 2, a laccase from Panus rudis was successfully expressed and purified using Pichia pastoris to obtain a highly active laccase for further downstream lignin degradation assays; however, only a low yield of protein was obtained. Owing to the inherent complexity of lignin, fractionation methodologies were utilised in Chapter 3 to simplify a frequently used, commercially available lignin into more uniform, lower molecular weight fractions for use in streamlined downstream depolymerisation. Uniformity was determined using nuclear magnetic resonance spectroscopy (NMR), which quantified β-O-4 linkage content, whilst Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) allowed the characterisation of the chemical diversity, identifying the presence of valuable monolignols. Chapter 4 is concerned with the characterisation of industrially relevant lignin fractions supplied by MetGen Oy, including the use of FT-ICR MS to characterise and compare the chemical diversity of an enzymatically fractioned lignin. This provided a basis for further enzymatic depolymerisation experiments. Subsequently, Chapter 5 focused on the treatment of a lignin fraction with an alkaliphilic laccase provided by MetGen Oy. FT-ICR MS highlighted the enzymatic targeting of compounds such as syringaresinol, which was almost wholly depleted after 24 hours, with NMR spectroscopy also indicating a >50% loss of β-β linkages. Ultimately this work aims to aid future developments in lignin research, by outlining strategies to improve, analyse and track the enzymatic depolymerisation of lignin.