Purification and characterization of Aspergillus parasiticus cytosine deaminase for possible deployment in suicide gene therapy
نویسندگان
چکیده
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 ̊C 45 ̊C, activation energy (Ea) of 8.4 KJ/mol and a t1/2 of 1.10 hr. A. parasiticus CD stoichiometrically deaminated Cytosine and 5-fluorocytosine (5-FC) with the KM values of 0.19 mM and 0.30 mM respectively. Studies on the effect of pH on KM and Vmax 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 Ca, Co, Zn and Hg and completely inhibited by Cu and Fe at 50 mM. Therefore, A. parasiticus CD can be compared with CDs from other sources that are used in suicide gene therapy.
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Evaluation of Thermostable Yeast Cytosine Deaminase and Herpes Simplex Virus Thymidine Kinase Fusions in Double Suicide Gene Therapy for Cancer
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