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Influence of cofactor change on the biochemical characteristics of a metalloendopeptidase isolated from Trametes coccinea (Tc-LysN) and expressed in Pichia pastoris

  • Metalloendopeptidases belong to a class of enzymes that digest proteins dependently on a divalent metal ion as a cofactor, which is usually zinc. Metalloendopeptidases can be specific for the N-terminus or C-terminus of different amino acids. For instance, the Peptidyl-Lys class is specific for the amino acid lysine and various peptidases were previously isolated from the fruiting bodies of fungi,Metalloendopeptidases belong to a class of enzymes that digest proteins dependently on a divalent metal ion as a cofactor, which is usually zinc. Metalloendopeptidases can be specific for the N-terminus or C-terminus of different amino acids. For instance, the Peptidyl-Lys class is specific for the amino acid lysine and various peptidases were previously isolated from the fruiting bodies of fungi, such as Grifola frondosa, Pleurotus ostreatus, and Armillaria mellea (Zhao et al., 2020). The previously discovered metalloendopeptidases specific for lysine were active at an optimum alkaline pH, however the recently isolated Tc-LysN is an endopeptidase which has a pH optimum in acidic conditions. Tc-LysN is a peptidyl-lys metalloendopeptidase, an endopeptidase specific for the N-terminus of lysine. Its resistance to high temperature and its activity optimum at acidic pH (Ahmed et al., 2024) makes it a valuable option for proteomic experiments. The peptidase used in this Master Thesis was genetically engineered in P. pastoris by Uzair et al. Initially, the P. pastoris mutant was cultivated in shake flasks with different growing conditions, looking for an increased titer of the protein. Afterward, the protein was produced and isolated with a single step anion-exchange chromatography at pH 7.2. After the peptidase was purified, its apoenzyme was originated using the chelating agent 1,10-phenantroline, which successfully inhibited the enzyme. Subsequently, the apoenzyme was reactivated with Zn2+ and Co2+. The reactivated apoenzymes achieved diverse biochemical characteristics, compared to the native enzyme, presenting an increased enzyme activity of 110% for the Co2+ reactivated apoenzyme, although paired with less specificity. Furthermore, also a reduction of temperature resistance was recorded, where after 1 hour of incubation at 70 °C less than 10% activity was recorded. Finally, the studies on this kind of peptidases are important because of their possible pivotal role in proteomic experiments (Zhao et al., 2020). The modification of the biochemical characteristics of these peptidases could give important tools that could be used in future applications.show moreshow less

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Metadaten
Document Type:Master's Thesis
Zitierlink: https://opus.hs-offenburg.de/8889
Bibliografische Angaben
Title (English):Influence of cofactor change on the biochemical characteristics of a metalloendopeptidase isolated from Trametes coccinea (Tc-LysN) and expressed in Pichia pastoris
Author:Matteo Albuge
Advisor:Thomas Eisele, Uzair Ahmed
Year of Publication:2024
Publishing Institution:Hochschule Offenburg
Granting Institution:Hochschule Offenburg
Place of publication:Offenburg
Publisher:Hochschule Offenburg
Page Number:V, 40
Language:English
Inhaltliche Informationen
Institutes:Fakultät Maschinenbau und Verfahrenstechnik (M+V)
Collections of the Offenburg University:Abschlussarbeiten / Master-Studiengänge / MBT
DDC classes:500 Naturwissenschaften und Mathematik
GND Keyword:Biochemie
Tag:LysN; Metalloendopeptidase; Trametes Coccinea
Formale Angaben
Open Access: Closed 
Licence (German):License LogoUrheberrechtlich geschützt