Fig. 1: Activity-based protein profiling (ABPP) for identifying lignocellulose-degrading biocatalysts from P. chrysosporium.

a Overview on the ABPP workflow for identifying lignocellulose-degrading enzymes from P. chrysosporium suspension cultures grown on minimal medium with beech wood chips. An ABP is added to either the filtrate of a P. chrysosporium beech wood culture (denoted as supernatant) or the dodecylmaltoside-solubilized substrate-bound fraction (denoted as SBF) after lyophilization. The pretreatment is followed by a standard ABPP workflow, consisting of click-attachment of a biotin-residue for affinity enrichment (in case of a two-step ABP), affinity enrichment of labeled enzymes, trypsin digest, and subsequent MS-based protein identification. The use of enzyme class-specific ABPs, therefore, results in targeted identification of active biocatalysts and thus functional enzyme screening and also enables the sequence-independent identification of novel biocatalysts without similarity to known homologous. The inlet image shows how P. chrysosporium binds to the solid wood surface during lignocellulose degradation. b Chemical structures of the ABPs and competitors used in this study. These are FP-alkyne (the “classical” serine hydrolase ABP) and JJB111 (GH ABP), as well as the FP competitor paraoxon and the JJB111 competitors KY371 and KY358. c Lignocellulose is a complex and recalcitrant polymer built up from cellulose, xylan (hemicellulose), and lignin. Its degradation requires the synergistic action of various different enzymes.