Purification and Characterization of Ornithine Decarboxylase from Aspergillus terreus; Kinetics of Inhibition by Various Inhibitors

Abstract

l-Ornithine decarboxylase (ODC) is the rate-limiting enzyme of de novo polyamine synthesis in humans and fungi. Elevated levels of polyamine by over-induction of ODC activity in response to tumor-promoting factors has been frequently reported. Since ODC from fungi and human have the same molecular properties and regulatory mechanisms, thus, fungal ODC has been used as model enzyme in the preliminary studies. Thus, the aim of this work was to purify ODC from fungi, and assess its kinetics of inhibition towards various compounds. Forty fungal isolates were screened for ODC production, twenty fungal isolates have the higher potency to grow on L-ornithine as sole nitrogen source. Aspergillus terreus was the most potent ODC producer (2.1 µmol/mg/min), followed by Penicillium crustosum and Fusarium fujikuori. These isolates were molecularly identified based on their ITS sequences, which have been deposited in the NCBI database under accession numbers MH156195, MH155304 and MH152411, respectively. ODC was purified and characterized from A. terreus using SDS-PAGE, showing a whole molecule mass of ~110 kDa and a 50 kDa subunit structure revealing its homodimeric identity. The enzyme had a maximum activity at 37 °C, pH 7.4–7.8 and thermal stability for 20 h at 37 °C, and 90 days storage stability at 4 °C. A. terreus ODC had a maximum affinity (Km) for l-ornithine, l-lysine and l-arginine (0.95, 1.34 and 1.4 mM) and catalytic efficiency (kcat/Km) (4.6, 2.83, 2.46 × 10−5 mM−1·s−1). The enzyme activity was strongly inhibited by DFMO (0.02 µg/mL), curcumin (IC50 0.04 µg/mL), propargylglycine (20.9 µg/mL) and hydroxylamine (32.9 µg/mL). These results emphasize the strong inhibitory effect of curcumin on ODC activity and subsequent polyamine synthesis. Further molecular dynamic studies to elucidate the mechanistics of ODC inhibition by curcumin are ongoing.

Biochemical Properties of Tyrosinase from Aspergillus terreus and Penicillium copticola; Undecanoic Acid from Aspergillus flavus, an Endophyte of Moringa oleifera, Is a Novel Potent Tyrosinase Inhibitor

“…The morphological identities of the potent fungal isolates Aspergillus terreus and Penicillium copticola were confirmed from the molecular authentication based on their ITS sequences. Similar screening paradigm for enzymes production by fungi were reported [27,42]. Coincidently, different tyrosinase producing fungal isolates have been documented [1,2,17].…”

“…After incubation for 7 days at 30 °C, the fungal mycelial pellets were collected, and washed by Tris-HCl (pH 7.0, 5 mM). Five grams of the fungal fresh weight were pulverized in liquid nitrogen, dispensing in Tris-HCl (pH 7.0, 5 mM) of 1 mM EDTA, 1 mM PMSF and 1 mM DTT [ 26 , 27 , 28 ]. The mixture was vortexed for 5 min, then centrifuged at 8000 rpm for 10 min at 4 °C, and the supernatant was used as the crude source for intracellular enzymes.…”

Exploiting the Biosynthetic Potency of Taxol from Fungal Endophytes of Conifers Plants; Genome Mining and Metabolic Manipulation

“…So many natural compounds are reported to use as antibiotics, anticancer agents. Many endophytic fungi isolated from different medicinal plants are reported to be main source of many secondary metabolites and pharmaceutical compounds with biotechnological roles [ 3 , 11 , 12 , 13 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 32 , 34 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 78 , 79 , 97 , 99 , 100 , 101 , 102 , 103 , 104 ]. Fusarubin and anhydrofusarubin, isolated from the Cladosporium species an endophyte of Rauwolfia serpentina , displayed a promising antimicrobial and antitumor activities [ 105 ].…”

Biosystematic Study on Some Egyptian Species of Astragalus L. (Fabaceae)

“…Distance estimates were performed by unweighted pair-group method analysis using arithmetic averages (UPGMA). The total genomic DNA of Astragalus L. was extracted from fresh young leaves of plants with CTAB (Cetyl trimethyl ammonium bromide) lysis buffer [43][44][45][46][47][48][49][50][51][52]. Brief description to CTAB method (three-five sentences).…”