Activity-based protein profiling assay for functional biocatalyst screening in white rot fungus Phanerochaete chrysosporium grown on beech wood chips

Activity-based protein profiling (ABPP) has emerged as a versatile biochemical method for studying enzyme activity under various physiological conditions, with applications mainly in the field of biomedicine. Here, we show the potential of ABPP in the discovery of biocatalysts from Phanerochaete chrysosporium suspension cultures grown on beech wood chips. P. chrysosporium is a thermophilic white rot fungus that expresses a large enzyme repertoire for efficient lignocellulose hydrolysis. By employing a comparative ABPP approach, we could preselect lignocellulose-degrading serine and glycoside hydrolases from this enzyme repertoire for further biochemical characterization. In addition to the supernatant, also a substrate-bound protein fraction isolated from wood chips was screened for active enzymes. As heterologous expression of fungal enzymes remains challenging, our ABPP-mediated preselection procedure allows to focus experimental efforts on the most promising biocatalysts. Besides, we show that this approach can also be used to functionally annotate domains-of-unknown functions (DUFs). ABPP-based biocatalyst screening may thus allow the elucidation of biocatalysts with novel gene sequences, and we anticipate that the presented ABPP approach will find wider applications both in the discovery of novel biocatalysts and in revealing the catalytic activity of hitherto unknown enzymes.