Purification and Characterization of Thermostable Cytosine Deaminase from Aspergillus fumigatus
- 21 February 2021
- journal article
- research article
- Published by Egypts Presidential Specialized Council for Education and Scientific Research in Egyptian Journal of Botany
- Vol. 61 (2), 479-490
- https://doi.org/10.21608/ejbo.2021.57312.1606
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 applicationsKeywords
This publication has 20 references indexed in Scilit:
- Nitrogen regulation of fungal secondary metabolism in fungiFrontiers in Microbiology, 2014
- Rationally designed Escherichia coli cytosine deaminase mutants with improved specificity towards the prodrug 5-fluorocytosine for potential gene therapy applicationsMedChemComm, 2012
- Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for FungiProceedings of the National Academy of Sciences of the United States of America, 2012
- Bacterial Cytosine Deaminase Mutants Created by Molecular Engineering Show Improved 5-Fluorocytosine–Mediated Cell Killing In vitro and In vivoCancer Research, 2009
- Recent Advances in Nitrogen Regulation: a Comparison between Saccharomyces cerevisiae and Filamentous FungiEukaryotic Cell, 2008
- Yeast Cytosine Deaminase Mutants with Increased Thermostability Impart Sensitivity to 5-FluorocytosineJournal of Molecular Biology, 2008
- Strong Enhancement of Recombinant Cytosine Deaminase Activity inBifidobacterium longumfor Tumor-Targeting Enzyme/Prodrug TherapyBioscience, Biotechnology, and Biochemistry, 2007
- Random mutagenesis and selection of Escherichia coli cytosine deaminase for cancer gene therapy"Protein Engineering, Design and Selection", 2004
- The 1.14 Å Crystal Structure of Yeast Cytosine Deaminase: Evolution of Nucleotide Salvage Enzymes and Implications for Genetic ChemotherapyStructure, 2003
- The structure of Escherichia coli cytosine deaminaseJournal of Molecular Biology, 2002