Purification, immobilization, and biochemical characterization ofl‐arginine deiminase from thermophilicAspergillus fumigatusKJ434941: Anticancer activityin vitro

Abstract

l-Arginine deiminase (ADI) has a powerful anticancer activity against various tumors, via arginine depletion, arresting the cell cycle at G1 phase. However, the current clinically tried bacterial ADI displayed a higher antigenicity and lower thermal stability. Thus, our objective was to purify and characterize this enzyme from thermophilic fungi, to explore its catalytic and antigenic properties for therapeutic uses. ADI was purified from thermophilic Aspergillus fumigatus KJ434941 to its electrophoretic homogeneity by 5.1-fold, with molecular subunit 50 kDa. The purified ADI was PEGylated and covalently immobilized on dextran to explore its catalytic properties. The specific activity of free ADI, PEG-ADI, and Dex-ADI was 26.7, 21.5, and 18.0 U/mg, respectively. At 50°C, PEG-ADI displays twofold resistance to thermal denaturation (t1/2 13.9 h), than free ADI (t1/2 6.9 h), while at 70°C, the thermal stability of PEG-ADI was increased by 1.7-fold, with similar stability to Dex-ADI with the free one. Kinetically, free ADI had the higher catalytic affinity to arginine, followed by PEG-ADI and Dex-ADI. Upon proteolysis for 30 min, the residual activity of native ADI, PEG-ADI, and Dex-AD was 8.0, 32.0, and 20.0% for proteinase K and 10.0, 52.0, and 90.0% for acid protease, respectively. The anticancer activity of the ADIs was assessed against HCT, HEP-G2, and MCF7, in vitro. The free and PEG-ADI exhibits a similar cytotoxic efficacy for the tested cells, lower than Dex-ADI. The free ADI had IC50 value 22.0, 16.6, and 13.9 U/mL, while Dex-ADI had 3.98, 5.18, and 4.43 U/mL for HCT, MCF7, and HEPG-2, respectively. The in vitro anticancer activity of ADI against HCT, MCF7, and HEPG-2 was increased by five-, three-, and threefold upon covalent modification by dextran. The biochemical and hematological parameters of the experimented animals were not affected by ADIs dosing, with no signs of anti-ADI immunoglobulins in vivo. The in vivo half-life time of free ADI, PEG-ADI, and Dex-ADI was 29.7, 91.1, 59.6 h, respectively. The present findings explored a novel thermostable, less antigenic ADI from thermophilic A. fumigatus, with further molecular and crystallographic analyses, this enzyme will be a powerful candidate for clinical trials.