A novel, robust peptidyl-lys metalloendopeptidase from Trametes coccinea recombinantly expressed in Komagataella phaffii

• DOI: 10.1007/s00253-023-12986-3
• Publication Date: 2024-01-13T09:02:51Z
• Authors: Uzair Ahmed; Tobias Stadelmann; Daniel Heid; Berit Würtz; Jens Pfannstiel; Katrin Ochsenreither; Thomas Eisele
• Keywords: Thermostability; Cyclic peptide

Abstract A novel peptidyl-lys metalloendopeptidase ( Tc -LysN) from Tramates coccinea was recombinantly expressed in Komagataella phaffii using the native pro-protein sequence. The peptidase secreted into culture broth as zymogen (~38 kDa) and mature enzyme (~19.8 simultaneously. -LysN purified to homogeneity with a single step anion-exchange chromatography at pH 7.2. N-terminal sequencing TMTpro Zero mass spectrometry of Tc- LysN indicated that pro-peptide cleaved between amino acid positions 184 185 Kex2 cleavage site present optimum determined be 5.0 while it maintained ≥60% activity values 4.5—7.5 ≥30% 8.5—10.0, indicating its broad applicability. temperature maximum 60 °C. After 18 h incubation 80 °C, still retained ~20% activity. Organic solvents such methanol acetonitrile, concentrations high 40% (v/v), were found enhance -LysN’s up ~100% ~50%, respectively. thermostability, ability withstand 8 M urea, tolerance organic solvents, an acidic make viable candidate employed proteomics workflows which alkaline conditions might pose challenge. nano-LC-MS/MS analysis revealed bovine serum albumin (BSA)’s sequence coverage 84% comparable 90% by trypsin peptides. Key points • Trametes (Tc-LysN) Tc-LysN is thermostable, applicable over range, tolerates denaturants successfully applied for protein digestion fingerprinting