Characterising peptidase activity of barley endoprotease B in complex technical matrices of the brewing process

Barley cysteine endoprotease B (HvEPB) is an important enzyme in plant biology and food production. Pilot-scale fed-batch fermentation with Komagataella phaffii in minimal FM22 medium enabled controlled recombinant HvEPB (r-HvEPB) secretion. Peptidomics across four technological brewing matrices - raw barley, malt, wort and beer - revealed hydrophobic P2 preference (V, L, Y) and heterogeneous P1 selection (T, Q, G), with certain residues disfavoured (S, A, G at P2; P, I, L at P1). Cleavage motifs shifted depending on the underlying matrix, primarily reflecting differences in substrates properties and availability. Immunogenic gluten peptides, particularly from C- and B-hordeins, were efficiently cleaved. Enzyme-linked immunosorbent assay confirmed 85-89% gluten reduction across all four matrices under controlled in vitro incubation. r-HvEPB hydrolysed brewing-relevant hordeins, α/β-amylases, while LTP I and serpin Z4 were less cleaved. Weighted motif analysis and inhibition trials underscored HvEPB's pivotal role as a broad-specificity endoprotease in malt-based systems, with r-HvEPB showing significantly higher gluten hydrolysis than endogenous malt proteases.