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

  • 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-terminalA 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.show moreshow less

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Metadaten
Document Type:Article (reviewed)
Zitierlink: https://opus.hs-offenburg.de/8266
Bibliografische Angaben
Title (English):A novel, robust peptidyl-lys metalloendopeptidase from Trametes coccinea recombinantly expressed in Komagataella phaffii
Author:Uzair AhmedStaff Member, Tobias StadelmannStaff Member, Daniel HeidStaff Member, Berit Würtz, Jens Pfannstiel, Katrin OchsenreitherORCiD, Thomas EiseleStaff MemberORCiDGND
Year of Publication:2024
Date of first Publication:2024/01/13
Publisher:SpringerNature
First Page:1
Last Page:12
Parent Title (English):Applied Microbiology and Biotechnology
Volume:108
ISSN:0175-7598 (Print)
ISSN:1432-0614 (Elektronisch)
DOI:https://doi.org/10.1007/s00253-023-12986-3
URN:https://urn:nbn:de:bsz:ofb1-opus4-82665
Language:English
Inhaltliche Informationen
Institutes:Fakultät Maschinenbau und Verfahrenstechnik (M+V)
Collections of the Offenburg University:Bibliografie
Tag:Acidic endopeptidase; Disulfide mapping; Kex2; Lys-N; Maturation; Peptidyl-lys metalloendopeptidase; Proteomics; Trypsin; Zymogen
Formale Angaben
Relevance for "Jahresbericht über Forschungsleistungen":Wiss. Zeitschriftenartikel reviewed: Listung in Master Journal List
Open Access: Open Access 
 Gold 
Licence (German):License LogoCreative Commons - CC BY - Namensnennung 4.0 International