Biochemical and Pharmacokinetic Properties of PEGylated Cystathionine &#947;-Lyase from <b><i>Aspergillus carneus</i></b> KF723837

Abstract

Cystathionine γ-lyase (CGL) was purified to its electrophoretic homogeneity from Aspergillus carneus by various chromatographic approaches. The purified enzyme has four identical subunits of 52 kDa based on SDS and native PAGE analyses. To improve its structural stability, purified CGL was modified by covalent binding to polyethylene glycol moieties. The specific activity of free-CGL and PEG-CGL was 59.71 and 48.71 U/mg, respectively, with a PEGylation yield of 81.5 and 70.7% modification of surface ε-amino groups. Free- and modified CGL have the same pattern of pH stability (8.0-9.0). At 50°C, the thermal stability [half-life time (T_1/2)] of PEG-CGL was increased by 40% in comparison to free-CGL. The activity of CGL was completely inhibited by hydroxylamine and Hg⁺², with no effect by EDTA. Free-CGL (0.04 m<smlcap>M</smlcap>^-1s^-1) and PEG-CGL (0.03 m<smlcap>M</smlcap>^-1s^-1) have a similar catalytic efficiency to <smlcap>L</smlcap>-cystathionine as a substrate. The inhibition constant values of propargylglycine were 0.31 and 0.52 µ<smlcap>M</smlcap> for the free- and PEG-CGL, respectively. By in vitro proteolysis, PEG-CGL retains >50% of its initial activity compared to <10% of the free-CGL for acid protease for 30 min. From in vivo pharmacokinetics in New Zealand white rabbits, the T_1/2 was 19.1 and 28.9 h for the Holo free-CGL and PEG-CGL, respectively, ensuring the role of PEGylation on shielding the CGL surface from proteolytic attack, reducing its antigenicity, and stabilizing its internal Schiff base. By external infusion of pyridoxal 5′-phosphate (10 µ<smlcap>M</smlcap>), the T_1/2 of free- and PEG-CGL was prolonged to 24 and 33 h, respectively, so dissociation of pyridoxal 5′-phosphate was one of the main causes of loss of enzyme activity. The biochemical and hematological responses of rabbits to free- and PEG-CGL were assessed, with relative similarity to the negative control, confirming the nil toxicity of enzymes. The titer of IgG was duplicated in response to free- versus PEG-CGL after 45 days. To the best of our knowledge, this is the first report concerned with purification and PEGylation of CGL from fungi, with higher affinity for <smlcap>L</smlcap>-cystathionine. With further molecular studies, CGL will be a promising enzyme against various cardiovascular diseases and antioxidant deficiency, as well as for generation of a neurotransmitter (H₂S).

Production, bioprocess optimization and γ-irradiation of Penicillium polonicum, as a new Taxol producing endophyte from Ginko biloba

“…The recovered fungal isolates were identified to their species levels based on their micro and micro-morphological features by growing on Czapek’s-Dox and PDA media for 10 days at 30 °C, according to the references key [ 28 , 29 ]. The highest potential Taxol producing fungal isolates were further molecularly identified based on the sequence of internal transcribed spacer (ITS) flanking the 5.8S region (ITS1-5.8S-ITS2 rDNA) [ 16 , 30 , 31 ]. Fungal genomic DNA (gDNA) was extracted by pulverizing the mycelia (∼0.2 g) in liquid nitrogen then adding 1 ml cetyltrimethylammonium bromide (CTAB) extraction buffer (2% CTAB, 2% PVP40, 0.2 % 2-mercaptoethanol, 20 mM EDTA, 1.4 M NaCl in 100 mM Tris-HCl, pH 8.0).…”

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

“…The activity and concentration of ODC were determined for each fraction as described above. The molecular homogeneity of the active fractions was assessed by SDS-PAGE, the most active and molecular homogenous fractions were gathered, downstream purified by gel-filtration chromatography by Sephadex G100 column [67]. The enzyme was loaded into the pre-equilibrated column, and eluted by PP buffer containing 10 µM PLP.…”

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 ].…”

“…The current approaches for Taxol production are; 1- Natural sources from the bark of T. brevifolia (most productive source), however, the yield of taxol based on this approach was ranged from 0.001–0.05%, thus for producing of one gram purified Taxol it need about 8–10 kg of plant bark, which collected from about 4 to 5 plants [ 9 ]. However, the scarce availability and higher vulnerability of this plant to unpredicted fluctuation with the ecological and environmental conditions [ 10 ] are the challenges for this source [ 3 , 11 , 12 , 13 ]. 2-Semisynthetic process via 10-decaetylbaccatin III intermediate from the needles of T. baccata is the current approach for taxol production [ 14 , 15 ], however, the lower yield of this intermediate, selectivity over unwanted byproducts, heterogeneity, reproducibility, in addition to the epigenetic and mutational changes of T. baccata are the current hurdles [ 16 , 17 ].…”