Plasma pharmacokinetics and tissue distribution of L-lysine α-oxidase from Trichoderma cf. aureoviride RIFAI VKM F- 4268D in mice.

L-lysine α-oxidase (LO) is an L-amino acid oxidase with antitumor, antimicrobial and antiviral properties. Pharmacokinetic (PK) studies were carried out by measuring LO concentration in plasma and tissue samples by enzyme immunoassay. L-lysine concentration in samples was measured spectrophotometrically using LO. After single i.v. injection of 1.0, 1.5, 3.0 mg/kg the circulating T1/2 of enzyme in mice varied from 51 to 74 min and the AUC0-inf values were 6.54 ± 0.46, 8.66 ± 0.59, 9.47 ± 1.45 µg/ml × h, respectively. LO was distributed in tissues and determined within 48 h after administration with maximal accumulation in liver and heart tissues. Mean time to reach the maximum concentration was highest for the liver-9 h, kidney-1 h and 15 min for the tissues of heart, spleen and brain. T1/2 of LO in tissues ranged from 7.75 ± 0.73 to 26.10 ± 2.60 h. In mice, plasma L-lysine decreased by 79% 15 min after LO administration in dose 1.6 mg/kg. The serum L-lysine levels remained very low from 1 to 9 h (< 25 µM, 17%), indicating an acute lack of L-lysine in animals for at least 9 h. Concentration of L-lysine in serum restored only 24 h after LO administration. The results of LO PK study show that it might be considered as a promising enzyme for further investigation as a potential anticancer agent.

[Synthesis of L-lactate oxidaze in yeast Yarrowia lipolytica during submerged cultivation].

The biosynthesis of L-lactate oxidase in the Yarrowia lipolytica yeast during submerged cultivation in laboratory bioreactors ANKUM-2M has been studied. It has been shown under optimal conditions of yeast cultivation with L-lactate that 24.5 U/L enzyme accumulated in the medium and the yield was 2.0 U/(L h). An increase in the biosynthesis of L-lactate oxidase to 75 U/L and the yield to 3.2 U/(L h) was achieved in the medium with L-lactate (1%) and glucose (2%). The enzyme was purified 251 times to homogeneity by hydrophobic and ion exchange chromatography state with a yield of 45% and a specific activity of 55.3 U/mg. Techniques of gel filtration and denaturing electrophoresis showed that L-lactate oxidase from Y. lipolytica is a tetramer with a molecular mass of 200­230 kDa. The enzyme showed a strict specificity to L-lactate and did not oxidize fumarate, pyruvate, succinate, ascorbate, dihydroxyacetone, glycolate, D-lactate, D, L-2-hydroxybutyrate and D, L-alanine or D-serine.

[Antistress systems of the yeast Yarrowia lipolitica (review)].

The authors' and literature data on the adaptation response of the micromycetes Yarrowia lipolytica to various stress impacts are considered in the review. The uniformity of cellular response to all stress factors is discussed.

[Effect of L-lysine alpha-oxidase from Trichoderma cf. aureoviride Rifai ВКМF-4268D on pheochromocytoma PC12 cell line].

L-Amino acid oxidases (L-ААО, EC 1.4.3.2) comprise a group of flavoproteins, catalyzing oxidative deamination of L-alpha amino acids to the corresponding alpha-keto acids, NH3 and Н2О2. In most cases these enzymes present homodimeric molecules with a molecular mass of 100-150 kDa, which were shown to possess antiviral, antifungal and antitumor activity. L-lysine alpha-oxidase (LO) holds an outstanding place among this group of enzymes and its biological role may differ significantly from the other L-AAO, because it cleaves an essential amino acid - L-lysine without significant action on the other amino acids. Although much research has examined LO effects in the organism, the molecular basis of these effects is yet to be identified. To fill this gap, the present work addressed one of hypothetical mechanisms of LO biological action using the enzyme from Trichoderma cf. aureoviride Rifai ВКМF-4268D and rat pheochromocytoma PC-12 as a model cell line. Using flow cytometry a dose-dependent cytotoxicity of LO was shown. The significant growth of intracellular reactive oxygen species levels, detected by 2,7-dichlorodihydrofluorescein assay, implies generation of peroxide as one of the molecular mechanisms of LO cytotoxic action, although this does not rule out other probable ways of LO action in the organizm.