Biochemical and Pharmacokinetic Properties of PEGylated Cystathionine γ-Lyase from Aspergillus carneus KF723837.

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 (T1/2)] 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 mM(-1)s(-1)) and PEG-CGL (0.03 mM(-1)s(-1)) have a similar catalytic efficiency to L-cystathionine as a substrate. The inhibition constant values of propargylglycine were 0.31 and 0.52 µM 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 T1/2 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 µM), the T1/2 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 L-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).

Screening, morphological and molecular characterization of fungi producing cystathionine γ-lyase.

The potency for production of cystathionine γ-lyase (CGL) by the fungal isolates was screened. Among the tested twenty-two isolates, Aspergillus carneus was the potent CGL producer (6.29 U/mg), followed by A. ochraceous (6.03 U/mg), A. versicolor (2.51 U/mg), A. candidus (2.12 U/mg), A. niveus and Penicillium notatum (2.0 U/mg). The potent six isolates producing CGL was characterized morphologically, A. carneus KF723837 was further molecularly characterized based on the sequence of 18S-28S rDNA. Upon sulfur starvation, the yield of A. carneus extracellular CGL was increased by about 1.7- and 4.1-fold comparing to non-sulfur starved and L-methionine free medium, respectively. Also, the uptake of L-methionine was duplicated upon sulfur starvation, assuming the activation of specific transporters for L-methionine and efflux of CGL. Also, the intracellular thiols and GDH activity of A. carneus was strongly increased by S starvation, revealing the activation of in vivo metabolic antioxidant systems. Upon irradiation of A. carneus by 2.0 kGy of γ-rays, the activity of CGL was increased by two-fold, regarding to control, with an obvious decreases on its yield upon further doses. Practically, CGL activity from the solid A. carneus cultures, using rice bran as substrate, was increased by 1.2-fold, comparing to submerged cultures, under optimum conditions.

Characterization of homocysteine γ-lyase from submerged and solid cultures of Aspergillus fumigatus ASH (JX006238).

Among 25 isolates, Aspergillus fumigatus ASH (JX006238) was identified as a potent producer of homocysteine gamma- lyase. The nutritional requirements to maximize the enzyme yield were optimized under submerged (SF) and solid-state fermentation (SSF) conditions, resulting in a 5.2- and 2.3-fold increase, respectively, after the last purification step. The enzyme exhibited a single homogenous band of 50 kDa on SDS-PAGE, along with an optimum pH of 7.8 and pH stability range of 6.5 to 7.8. It also showed a pI of 5.0, as detected by pH precipitation with no glycosyl residues. The highest enzyme activity was obtained at 37-40 degrees C, with a Tm value of 70.1 degrees C. The enzyme showed clear catalytic and thermal stability below 40 degrees C, with T1/2 values of 18.1, 9.9, 5.9, 3.3, and 1.9 h at 30 degrees C, 35 degrees C, 40 degrees C, 50 degrees C, and 60 degrees C, respectively. Additionally, the enzyme Kr values were 0.002, 0.054, 0.097, 0.184, and 0.341 S-1 at 30 degrees C, 35 degrees C, 40 degrees C, 50 degrees C, and 60 degrees C, respectively. The enzyme displayed a strong affinity to homocysteine, followed by methionine and cysteine when compared with non-S amino acids, confirming its potency against homocysteinuriarelated diseases, and as an anti-cardiovascular agent and a specific biosensor for homocysteinuria. The enzyme showed its maximum affinity for homocysteine (Km 2.46 mM, Kcat 1.39 × 10(-3) s(-1)), methionine (Km 4.1 mM, Kcat 0.97 × 10(-3) s(-1)), and cysteine (Km 4.9 m M, Kcat 0.77 × 10(-3) s(-1)). The enzyme was also strongly inhibited by hydroxylamine and DDT, confirming its pyridoxal 5'-phosphate (PLP) identity, yet not inhibited by EDTA. In vivo, using Swiss Albino mice, the enzyme showed no detectable negative effects on platelet aggregation, the RBC number, aspartate aminotransferase, alanine aminotransferase, or creatinine titer when compared with negative controls.

L-methioninase production by filamentous fungi: I-screening and optimization under submerged conditions.

Findings show 21 fungal isolates belonging to eight genera recovered from Egyptian soils that have the potential to attack L-methionine under submerged conditions. Aspergillus flavipes had the most methioninolytic activity, giving the highest yield of L-methioninase (10.78 U/mg protein), rate of methionine uptake (93.0%), and growth rate (5.0 g/l), followed by Scopulariopsis brevicaulis and A. carneus. The maximum L-methioninase productivity (11.60 U/mg protein) by A. flavipes was observed using L-methionine (0.8%) as an enzyme-inductive agent and glucose (1%) as a co-dissimilated carbon source. A significant reduction in L-methioninase biosynthesis by A. flavipes was detected using carbon-free medium, suggesting the lack of ability to use L-methionine as a carbon and nitrogen source. Potassium dihydrogen phosphate (0.25%), the best source of phosphorus, favors enzyme biosynthesis and enhances the level of methionine uptake by A. flavipes. The maximum L-methioninase productivity (12.58 U/mg protein) and substrate uptake (95.6%) were measured at an initial pH of 7.0.