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Egyptian Journal of Botany
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Volume Volume 62 (2022)
Volume Volume 61 (2021)
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El-Sayed, A., Ali, M., El-Sharkawy, R., El-sayed, N., Amer, M. (2021). Purification and Characterization of Thermostable Cytosine Deaminase from Aspergillus fumigatus . Egyptian Journal of Botany, 61(2), 479-490. doi: 10.21608/ejbo.2021.57312.1606
Ashraf El-Sayed; Mostafa G. Ali; Reyad El-Sharkawy; Nesma El-sayed; Mahmoud Amer. "Purification and Characterization of Thermostable Cytosine Deaminase from Aspergillus fumigatus ". Egyptian Journal of Botany, 61, 2, 2021, 479-490. doi: 10.21608/ejbo.2021.57312.1606
El-Sayed, A., Ali, M., El-Sharkawy, R., El-sayed, N., Amer, M. (2021). 'Purification and Characterization of Thermostable Cytosine Deaminase from Aspergillus fumigatus ', Egyptian Journal of Botany, 61(2), pp. 479-490. doi: 10.21608/ejbo.2021.57312.1606
El-Sayed, A., Ali, M., El-Sharkawy, R., El-sayed, N., Amer, M. Purification and Characterization of Thermostable Cytosine Deaminase from Aspergillus fumigatus . Egyptian Journal of Botany, 2021; 61(2): 479-490. doi: 10.21608/ejbo.2021.57312.1606

Purification and Characterization of Thermostable Cytosine Deaminase from Aspergillus fumigatus

Article 77, Volume 61, Issue 2, August 2021, Page 479-490  XML PDF (2.35 MB)
Document Type: Original Article
DOI: 10.21608/ejbo.2021.57312.1606
Authors
Ashraf El-Sayed email 1; Mostafa G. Ali email 1, 2; Reyad El-Sharkawy2; Nesma El-sayed2; Mahmoud Amer2
1Enzymology and Fungal Biotechnology Lab (EFBL), Botany and Microbiology Department, Faculty of Science, Zagazig University, Zagazig, 44519, Egypt
2Botany and Microbiology Department, Faculty of Science, Benha University, Benha, 13518, Egypt
Abstract
CYTOSINE Deaminase (CDA) is a non-human enzyme converting cytosine to uracil, with significant implementation on various cancer therapeutic approaches especially prodrug mediated therapy using 5-fluorocytosine into 5-fluorouracil. However, the lower catalytic/ thermal stability and higher antigenicity of this enzyme are the main challenges for further its clinical applications, thus, screening for thermostable enzyme with higher turnover activity was the objective of this study. Among the recovered thermotolerant fungal isolates, Aspergillus fumigatus has been selected as potent CDA producer. Upon nitrogen starvation, the yield of intracellular CDA by A. fumigatus was significantly increased by about 5 folds, comparing to control. The enzyme was purified from thermotolerant A. fumigates into its electrophoretic homogeneity by ion-exchange and gel-filtration chromatography by 4.4 purification folds and 29.9% yield, respectively, with molecular subunit structure 48 kDa under denaturing-PAGE. The purified enzyme showed an optimum pH 7.0, optimum reaction temperature 37°C. The maximum affinity (Km) and reaction velocity (Vmax) of purified CDA was 0.08 mM and 400 μmol/min/mg on cytosine as substrate. At 37°C, the half-life time (T1/2) of purified CDA was about 8 h, ensuring the structural/ catalytic thermal stability of this enzyme. Based on these preliminary results, A. fumigatus CDA could be a scaffold for further in vivo studies on cancer prodrug-mediated therapies, or gene therapy applications.
Keywords
Aspergillus fumigatus; Cytosine; Cytosine deaminase; 5-Fluorocytosine; Purification
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