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Article
2024
A novel, robust peptidyl-lys metalloendopeptidase from Trametes coccinea recombinantly expressed in Komagataella phaffii
A novel, robust peptidyl-lys metalloendopeptidase from Trametes coccinea recombinantly expressed in Komagataella phaffii
Abstract (English)
A novel peptidyl-lys metalloendopeptidase ( Tc -LysN) from Tramates coccinea was recombinantly expressed in Komagataella phaffii using the native pro-protein sequence. The peptidase was secreted into the culture broth as zymogen (~38 kDa) and mature enzyme (~19.8 kDa) simultaneously. The mature Tc -LysN was purified to homogeneity with a single step anion-exchange chromatography at pH 7.2. N-terminal sequencing using TMTpro Zero and mass spectrometry of the mature Tc- LysN indicated that the pro-peptide was cleaved between the amino acid positions 184 and 185 at the Kex2 cleavage site present in the native pro-protein sequence. The pH optimum of Tc -LysN was determined to be 5.0 while it maintained ≥60% activity between pH values 4.5—7.5 and ≥30% activity between pH values 8.5—10.0, indicating its broad applicability. The temperature maximum of Tc -LysN was determined to be 60 °C. After 18 h of incubation at 80 °C, Tc -LysN still retained ~20% activity. Organic solvents such as methanol and acetonitrile, at concentrations as high as 40% (v/v), were found to enhance Tc -LysN’s activity up to ~100% and ~50%, respectively. Tc -LysN’s thermostability, ability to withstand up to 8 M urea, tolerance to high concentrations of organic solvents, and an acidic pH optimum make it a viable candidate to be employed in proteomics workflows in which alkaline conditions might pose a challenge. The nano-LC-MS/MS analysis revealed bovine serum albumin (BSA)’s sequence coverage of 84% using Tc -LysN which was comparable to the sequence coverage of 90% by trypsin peptides.
Key points
• A novel LysN from Trametes coccinea (Tc-LysN) was expressed in Komagataella phaffii and purified to homogeneity • Tc-LysN is thermostable, applicable over a broad pH range, and tolerates high concentrations of denaturants • Tc-LysN was successfully applied for protein digestion and mass spectrometry fingerprinting
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Applied microbiology and biotechnology, 108 (2024), 1.
https://doi.org/10.1007/s00253-023-12986-3.
ISSN: 1432-0614
Berlin/Heidelberg : Springer
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Ahmed, U., Stadelmann, T., Heid, D., Würtz, B., Pfannstiel, J., Ochsenreither, K., & Eisele, T. (2024). A novel, robust peptidyl-lys metalloendopeptidase from Trametes coccinea recombinantly expressed in Komagataella phaffii. Applied microbiology and biotechnology, 108. https://doi.org/10.1007/s00253-023-12986-3
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@article{Ahmed2024,
doi = {10.1007/s00253-023-12986-3},
author = {Ahmed, Uzair and Stadelmann, Tobias and Heid, Daniel et al.},
title = {A novel, robust peptidyl-lys metalloendopeptidase from Trametes coccinea recombinantly expressed in Komagataella phaffii},
journal = {Applied Microbiology and Biotechnology},
year = {2024},
volume = {108},
}