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CYP116B5‐SOX: An artificial peroxygenase for drug metabolites production and bioremediation

Articolo
Data di Pubblicazione:
2024
Abstract:
: CYP116B5 is a class VII P450 in which the heme domain is linked to a FMN and 2Fe2S-binding reductase. Our laboratory has proved that the CYP116B5 heme domain (CYP116B5-hd) is capable of catalyzing the oxidation of substrates using H2O2. Recently, the Molecular Lego approach was applied to join the heme domain of CYP116B5 to sarcosine oxidase (SOX), which provides H2O2 in-situ by the sarcosine oxidation. In this work, the chimeric self-sufficient fusion enzyme CYP116B5-SOX was heterologously expressed, purified, and characterized for its functionality by absorbance and fluorescence spectroscopy. Differential scanning calorimetry (DSC) experiments revealed a TM of 48.4 ± 0.04 and 58.3 ± 0.02°C and a enthalpy value of 175,500 ± 1850 and 120,500 ± 1350 cal mol-1 for the CYP116B5 and SOX domains respectively. The fusion enzyme showed an outstanding chemical stability in presence of up to 200 mM sarcosine or 5 mM H2O2 (4.4 ± 0.8 and 11.0 ± 2.6% heme leakage respectively). Thanks to the in-situ H2O2 generation, an improved kcat/KM for the p-nitrophenol conversion was observed (kcat of 20.1 ± 0.6 min-1 and KM of 0.23 ± 0.03 mM), corresponding to 4 times the kcat/KM of the CYP116B5-hd. The aim of this work is the development of an engineered biocatalyst to be exploited in bioremediation. In order to tackle this challenge, an E. coli strain expressing CYP116B5-SOX was employed to exploit this biocatalyst for the oxidation of the wastewater contaminating-drug tamoxifen. Data show a 12-fold increase in tamoxifen N-oxide production-herein detected for the first time as CYP116B5 metabolite-compared to the direct H2O2 supply, equal to the 25% of the total drug conversion.
Tipologia CRIS:
03A-Articolo su Rivista
Keywords:
H2O2 in‐situ generation; cytochrome P450; environmental pollution; molecular Lego; protein engineering
Elenco autori:
Giuriato, Daniele; Catucci, Gianluca; Correddu, Danilo; Nardo, Giovanna Di; Gilardi, Gianfranco
Autori di Ateneo:
CATUCCI Gianluca
CORREDDU Danilo
DI NARDO Giovanna
GILARDI Gianfranco
GIURIATO Daniele
Link alla scheda completa:
https://iris.unito.it/handle/2318/1976413
Link al Full Text:
https://iris.unito.it/retrieve/handle/2318/1976413/1296551/Biotechnology%20Journal%20-%202024%20-%20Giuriato%20-%20CYP116B5%BFSOX%20%20An%20artificial%20peroxygenase%20for%20drug%20metabolites%20production%20and.pdf
Pubblicato in:
BIOTECHNOLOGY JOURNAL
Journal
Progetto:
PRIN 2020 - SEAfood WAste Valorization by oxidative metalloEnzymes - Cda 24/02/2022
  • Dati Generali
  • Aree Di Ricerca

Dati Generali

URL

https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/10.1002/biot.202300664

Aree Di Ricerca

Settori (22)


LS1_12 - Protein design - (2022)

LS9_1 - Bioengineering for synthetic and chemical biology - (2022)

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PIANETA TERRA, AMBIENTE, CLIMA, ENERGIA e SOSTENIBILITA' - Energia e Fonti Energetiche

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