Amperometric flow-ikiection analytical system for quantitation of low L-lysine concentration was described. Llysine-a-oxidase from Trichoderme sp. was immobilized by covalent linking on silica gel. The ahift in oxygen concentration during enzymatic reaction was detected with a Clark membrane electrode. The rate of oxygen consumption was linearly dependent on Llysine concentration over the 0.20 -5,5 d. Response time was 14 s. the total assay the about 2 min. The influences of pH, ionic strength and nature of the buffer on enzyme catalytic activity were tested. The analysis conditions were optimized. The immobilized L-1yaine-U-oxidase retained the catalytic activity for about 5 months.
We studied the effects of a concentrate of metabolites of Trichoderma harzianum Rifai F-180, an active producer of L-lysine-α-oxidase, and homogenous enzyme on a highly virulent bacteria Erwinia amylovora. The producer of antitumor and antiviral Trichoderma enzyme L-lysine-α-oxidase was cultured on a processing system of G. K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences (Pushchino). Activity of L-lysine-α-oxidase in the prepared concentrate of metabolic products of the producer was 5.4 U/ml, and activity of the homogenous enzyme was 50 U/ml. Antibacterial activity of the enzyme was shown in our experiments.
Studies of the effects of Trichoderma harzianum Rifai F-180 culture fluid concentrate containing L-lysine-α-oxidase antitumor enzyme produced by the fungus and the homogenous enzyme, on ultrahazardous bacterium Acidovorax citrulli demonstrated the antibacterial activity of the concentrate. Trichoderma harzianum Rifai F-180 producing L-lysine-α-oxidase was cultured in a technological device at G. K. Skryabin Institute of Biochemistry and. Physiology of Microorganisms, Russian Academy of Sciences. Activity of L-lysine-α-oxidase in the resulted culture fluid concentrate was 0.54 U/ml, activity of the homogenous enzyme was 50 U/mg.
Positive effect of local treatment with L-lysine-a-oxidase on the course of herpes genital infection was demonstrated on guinea pigs even after the appearance of infection symptoms. In animals treated with L-lysine-a-oxidase, the severity of the disease, virus reproduction and virus-induced changes in cells were significantly less pronounced than in untreated animals, The preparations exerted no toxic effects. Key Words: herpes simplex virus type II," L-lysine-a-oxidaseL-lysine-a-oxidase (LO) is a promising antitumor enzyme. The growth-inhibiting effect of LO was first discovered by Japanese scientists and then confirmed by Russian researchers using a Trichoderma harzianum Rifai strain [1- [3,6].Herpetic infection often serves as an ethiological factor for cancer development. Our previous studies showed that LO exerts a powerful antiherpetic effect in vitro. The present study investigated the antiherpetic activity of LO in vivo. This article describes the effects of LO on genital herpes infection in guinea pigs. MATERIALS AND METHODSHerpes simplex virus type II (HSV-2), a MS strain (National Collection of Viruses of the USA) was used. It was maintained in sensitive Vero cells cultured in MEM medium containing 2% fetal calf serum and 500 U/ml benzylpenicillin.Infectious activity of viruses isolated from herpetic lesions was determined by a standard titer technique in Vero cell culture. The results were expressed in tissue cytopathic doses (TCDs0). Specific cytopathic effects of HSV-2 were verified by an immunofluorDepartment of Biochemistry, Russian State University of Peoples' Friendship, Moscow escent method using antiherpetic rabbit antibodies labeled with fluorescein isothiocyanate [4,5].Male guinea pigs weighing 300 g were intramuscularly anesthetized with ketalar (300 mg/ml) and infected with herpes by application penis of 0.1 ml virus suspension in a dose of 105 plague-forming units/ml corresponding 100% lethal dose on preliminary scarified. Clinical symptoms of acute genital herpes infection were assessed using a 5-point scale [7].Three groups were formed (15-20 animals each): group 1 (control) received no treatment; group 2 animals were daily treated with 0.5 ml LO gel (70 ~tg/ml) applied on herpetic lesion areas and given intramuscular injection of 0.25 ml LO (i00 ~g/ml); group 3 animals received only gel applications (0.5 ml, 70 lag/ml) on penis mucosa lesions.The treatment was started 1 or 48 h postinfection. The severity of viral infection was scored according to D. Mayo et al., [7] (0.5-1 points: erythema or 1-2 clusters of blisters; 2 points: 3-10 clusters of herpetic blisters; 3:11-20 clusters; 4: more than 21 clusters, fused blisters) and the mean score was calculated.
A method for PCR diagnosis of impatiens necrotic spot virus is developed. Concentrated culture fluid with active L-lysine-α-oxidase (0.54 U/ml) from Trichoderma harzianum Rifai fungus producer strain F-180 inhibits vitally hazardous impatiens necrotic spot phytovirus.
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