Purification and characterization of Aspergillus parasiticus cytosine deaminase for possible deployment in suicide gene therapy

Hassan Zanna, A. J. Nok, S. Ibrahim, H. M. Inuwa

Department Department of Biochemistry, University of Maiduguri, Maiduguri, Nigeria.
Department of Biochemistry, Ahmadu Bello University, Zaria, Nigeria.

DOI: 10.4236/abc.2012.22018                

Abstract

Cytosine Deaminase (CD) from Aspergillus parasiticus was purified and characterized. Time course for maximal CD production (50 μmol/min/mg) was at 72 hrs. The enzyme was purified 387.73 folds with an overall yield of 13%. The CD had pH optimum of 7.2, a temperature optimum of 40℃ - 45℃, activation of energy (Ea) of 8.4 KJ/mol and a t_1/2 of 1.10 hr. A. parasiticus CD stoichiometrically deaminated Cyto-sine and 5-fluorocytosine (5-FC) with the KM values of 0.19mM and 0.30 mM respectively. Studies on the effect of pH on KM and V_max gave pKa₁ of 5.8 and pKa₂ of 6.3 with enthalpy of ionization of 43.01 KJ/mole suggesting histidine in the active site. The enzyme was strongly inhibited by Ca²⁺, Co²⁺, Zn²⁺ and Hg²⁺ and completely inhibited by Cu²⁺ and Fe²⁺ at 50 mM. Therefore, A. parasiticus CD can be compared with CDs from other sources that are used in suicide gene therapy.

Keywords

Cytosine Deaminase; Cytosine, 5-Fluorocytosine; Aspergillus parasiticus; Activation Energy

Conflicts of Interest

The authors declare no conflicts of interest.

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