Biocatalysts (enzymes) are central to the development of a sustainable bioeconomy. However, to enable the successful scale-up of bio-catalysed processes, it is crucial to screen for and develop resilient enzymes that can work directly on industrial substrates, often under extreme process conditions. The focus of this work is the enzymatic functionalization of an abundant but complex bio-based feedstock, lignin. Despite its structural inertness, lignin is an attractive feedstock for industrial application1. Research on the enzymatic functionalization of industrially relevant lignins is limited, focusing on either simple activity screens with model compounds or the full degradation of lignin to monoaromatics. Herein, a screening pipeline has been developed, enabling the testing of enzymes with potential laccase-like activity directly on industrial lignin substrates while simultaneously comparing this with the activity on simpler, widely used compounds such as ABTS and 2,6 DMP. For testing activity directly on lignin, a modification of a method2 using solubilized/suspended lignins in a solid agarose-matrix is used, together with molecular-level probing using time-of-flight secondary-ion mass spectrometry (ToF-SIMS). A total of 10 fungal laccase candidates were recovered. The findings from this study demonstrate that activity on soluble lignin-like or standard synthetic substrates does not necessarily correlate with activity on industrial lignins. This pipeline allows for the recovery of robust enzymes that performed similarly on lignin, with or without purification, which is advantageous during scale-up. Lastly, all target sequences were analysed for similarity against fungal laccases previously characterised on lignin-like substrates by constructing sequence similarity networks. This analysis revealed 6 enzyme targets that were distinct from the characterised sequences, representing novel and resilient candidates.

Developing a screening pipeline to uncover resilient laccase enzyme candidates for surface-activation of industrial lignins

ACS Fall 2021

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