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.

Enhanced Plasma Persistence of Enzyme with Therapeutic Potential Using Polyethylene Glycol as a Coupling Agent

“…15,16 In the study by El-Sayed et al on ADI isolated from thermophilic Aspergillus fumigatus KJ434941, there was an overall three-fold increase in the plasma half-life of the enzyme upon pegylation. 17 The kinetic data of Km and Vmax values of PEGylated ADI was found to be 2.94 ± 0.13 mM and 129.87 ± 1.24 µmol/min, respectively. The Km value of the ADI was found to be slightly increased upon pegylation, which suggested that the attached PEG molecules on the surface of the enzyme did not significantly reduce the affinity for the substrate.…”

Optimization of Arginine Deaminase Production from Indigenous Bacterium Pseudomonas aeruginosa PS2

“…Among them ADI is one of the most important and best characterized enzymic drugs. The earlier application of ADI is focused against the treatment of hepatocellular carcinomas but in the recent past, scientific community trying to search new therapeutic applications of ADI in treatment of other arginine auxotrophic tumors such as pancreatic cancer (Liu et al, 2014), prostate cancer (Changou et al, 2014), leukemia (Miraki-Moud et al, 2015), colon cancer (El-Sayed et al, 2015), and breast cancer (Li et al, 2016). As we know that, ADI is a significant player of ADI or arginine dihydrolase (ADH) pathway and generating one molecule of ATP by phosphorylation of ADP.…”

Immobilization and Characterization of Purified Aspergillus flavus Peroxidase Mediated Silver Nanoparticle Synthesis: Peroxidase Surface Reactive Residues are Implemented for Reduction of Silver Ions, More than Its Active Sites

“…The crude peroxidase was fractionated by ammonium sulfate saturation (20-70%), incubation at 4°C for 2 h, then the mixture was centrifuged for 10 min at 4000 g. The precipitated protein was collected in potassium phosphate buffer containing 1 mM EDTA and dialyzed against the same buffer till complete removal of salt traces. The enzyme was purified by gel filtration chromatography using Sephadex G 100 and G 200 columns [26,[30][31][32]. After column equilibration with the same buffer, the sample was loaded to the column and the enzyme fractions were eluted at flow rate 0.2 ml/ min.…”

“…The mixture was centrifuged for 10 min at 5000 rpm, the activated chitosan was washed with distilled water, collected and suspended in 10 ml potassium phosphate buffer (pH 7.0). The glutaraldehyde-activated chitosan has free reactive aldehyde groups that form Schiff base with the reactive amino groups of peroxidase [32]. One ml of peroxidase (35.0 µmol/mg) was added to the 5 ml activated chitosan, incubated overnight at 4°Cwith gently shaking.…”