Aspergillus Niger thermostable Cytosine deaminase‑dextran conjugates with enhanced structure stability, proteolytic resistance, and Antiproliferative activity

Abstract

Cytosine deaminase (CDA) is a prodrug mediating enzyme converting 5-furocytosine into 5-furouracil with profound broad-range anticancer activity towards various cell lines. Availability, molecular stability, and catalytic efciency are the main limiting factors halting the clinical applications of this enzyme on prodrug and gene therapies, thus, screen‑ ing for CDA with unique biochemical and catalytic properties was the objective. Thermotolerant/ thermophilic fungi could be a distinctive repertoire for enzymes with afordable stability and catalytic efciency. Among the recovered thermotolerant isolates, Aspergillus niger with optimal growth at 45 °C had the highest CDA productivity. The enzyme was purifed, with purifcation 15.4 folds, molecular mass 48 kDa and 98 kDa, under denaturing and native PAGE, respectively. The purifed CDA was covalently conjugated with dextran with the highest immobilization yield of 75%. The free and CDA-dextran conjugates have the same optimum pH7.4, reaction temperature 37 °C, and pI 4.5, and similar response to the inhibitors and amino acids suicide analogues, ensuring the lack of efect of dextran conjuga‑ tion on the CDA conformational structure. CDA-Dextran conjugates had more resistance to proteolysis in response to proteinase K and trypsin by 2.9 and 1.5 folds, respectively. CDA-Dextran conjugates displayed a dramatic structural and thermal stability than the free enzyme, authenticating the acquired structural and catalytic stability upon dextran conjugation. The thermal stability of CDA was increased by about 1.5 folds, upon dextran conjugation, as revealed from the half-life time (T1/2). The afnity of CDA-conjugates (Km 0.15mM) and free CDA (Km 0.22mM) to deaminate 5-fuorocytosine was increased by 1.5 folds. Upon dextran conjugation, the antiproliferative activity of the CDA towards the diferent cell lines “MDA-MB, HepG-2, and PC-3” was signifcantly increased by mediating the prodrug 5-FC. The CDA-dextran conjugates strongly reduce the tumor size and weight of the Ehrlich cells (EAC), dramatically increase the titers of Caspase-independent apoptotic markers PARP-1 and AIF, with no cellular cytotoxic activity, as revealed from the hematological and biochemical parameters.