Assuntos
Aminoácido Oxirredutases/isolamento & purificação , Isoenzimas/isolamento & purificação , Trichoderma/enzimologia , Aminoácido Oxirredutases/química , Aminoácido Oxirredutases/efeitos dos fármacos , Catálise , Cromatografia por Troca Iônica , Focalização Isoelétrica , Isoenzimas/química , Isoenzimas/efeitos dos fármacos , Peso MolecularRESUMO
Homogeneous preparations of L-lysine-alpha-oxidase were obtained from Trichoderma sp cultivated by using a surface technique and a fermenter set. The homogeneous enzyme preparations were similar in molecular mass, isoelectric point and substrate specificity. There was the single difference in the absorbance spectra, which may occur due to the presence of cofactor FAD in various oxidation states. The findings suggest that cultivation of Trichoderma sp in the fermenter set did not alter properties of L-lysine-alpha-oxidase produced.
Assuntos
Aminoácido Oxirredutases/metabolismo , Trichoderma/enzimologia , Fermentação , Ponto Isoelétrico , Peso Molecular , Análise Espectral , Especificidade por SubstratoRESUMO
Some physicochemical properties of L-lysine oxidase from two strains of Trichoderma were studied. Content of metals and cofactors (Se, Zn, Cu, Fe, Co, Mn, Mo), amino acid analysis, secondary structure were estimated. The enzyme molecule from Trichoderma sp was found to contain both FAD and PQQ cofactors.
Assuntos
Aminoácido Oxirredutases/química , Trichoderma/enzimologia , Aminoácidos/análise , Cromatografia em Gel , Dicroísmo Circular , Flavina-Adenina Dinucleotídeo/química , Cofator PQQ , Quinolonas/química , Especificidade da Espécie , Espectrometria de FluorescênciaRESUMO
Regulation of biosynthesis of L-lysine-alpha-oxidase from Trichoderma sp using biostimulators was studied. Specific activity of L-lysine-alpha-oxidase was increased 2.7-fold in presence of biostimulators; this occurred due to induction of the enzyme, properties of which were described previously. Catalytic and physico-chemical properties of the enzyme studied were not altered after addition of stimulators into the cultivation medium of the producing strain. Small amounts of L-lysine-alpha-oxidase isozyme were detected in the medium during cultivation; the isozyme was less active and had lower molecular mass as compared with the enzyme studied.
Assuntos
Aminoácido Oxirredutases/biossíntese , Catálise , Eletroforese em Gel de Poliacrilamida , Indução Enzimática , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Especificidade por Substrato , Trichoderma/enzimologiaRESUMO
A new fungal strain, Trichoderma sp., discovered in Moscow, produces the antitumor enzyme, lysine-oxidase, which demonstrates an anti-invasive effect in vitro and anti-metastatic activity in vivo. Maximal inhibition of the in vitro invasion of MM1 clone cells was obtained when the tumor cells were pretreated with 2.5 mU/ml of lysine-oxidase; the pretreatment caused a 1.9-times reduction in cell growth and a 1.6-times reduction in the invasive capacity. We studied its anti-metastatic effect on the spreading Lewis lung carcinoma (3LL) in mice from which the primary tumor had been removed. The administration of the enzyme (50 U/kg, i.v.) significantly decreased not only the extent but the number of lung metastases, as compared with the untreated mice. In addition to that, the lysine-oxidase treatment considerably increases the life-span of mice from which the primary tumor had been removed (200 days after 3LL implantation, lysine-oxidase treatment caused surviving of 50% mice in experimental group).
Assuntos
Aminoácido Oxirredutases/uso terapêutico , Invasividade Neoplásica , Metástase Neoplásica/prevenção & controle , Trichoderma/enzimologia , Animais , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Neoplasias Experimentais/patologiaRESUMO
A simple and relatively sensitive procedure was developed for determination of L-lysine at 3-30 mmole/L concentration. The procedure does not involve the carcinogenic compound o-dianisidine. L-lysine alpha-oxidase catalyzed oxidative deamination of L-lysine with O2 consumption and formation of H2O2, NH3 and alpha-keto-epsilon-aminocaproic acid. Horseradish peroxidase and a non-carcinogenic compound 3,3,5,5'-tetramethylbenzidine dihydrochloride as an electron donor were used in determination of H2O2 formed. The procedure developed enabled also to measure the L-lysine alpha-oxidase activity at the enzyme concentrations of 10-500 ng/ml. The only limitation of the procedure is relatively low pH-values of the reaction medium.
Assuntos
Aminoácido Oxirredutases , Benzidinas , Lisina/análise , Trichoderma/enzimologia , Peroxidase do Rábano Silvestre/química , Peróxido de Hidrogênio/química , Concentração de Íons de Hidrogênio , Cinética , Análise EspectralRESUMO
A new procedure is developed for purification of the antitumoral enzyme L-lysyl-alpha-oxidase from Trichoderma sp. The procedure included two steps: hydrophobic chromatography on butyl sylochrome C-80 and chromatography using biospecific sorbent AH-Sepharose. The simplified procedure enabled to increase distinctly the specific enzymatic activity from 30 to 50 IU/mg in the preparation obtained with a good yield.
Assuntos
Aminoácido Oxirredutases/isolamento & purificação , Trichoderma/enzimologia , Cromatografia/métodosRESUMO
Evidences on the antileukemic effect of L-lysine alpha-oxidase from Trichoderma sp. are presented. It is inferred that enzyme needs detail studying as a potential chemotherapeutic drug both in experimental and clinical research.
Assuntos
Aminoácido Oxirredutases/uso terapêutico , Leucemia L1210/tratamento farmacológico , Leucemia P388/tratamento farmacológico , Leucemia Experimental/tratamento farmacológico , Fungos Mitospóricos/enzimologia , Trichoderma/enzimologia , Aminoácido Oxirredutases/administração & dosagem , Animais , Avaliação de Medicamentos , Masculino , Camundongos , Camundongos Endogâmicos , Fatores de TempoRESUMO
Relatively rapid procedure was developed for isolation and purification of the antitumor enzyme L-lysine alpha-oxidase. The procedure involved three steps instead of six or four steps described previously, with a yield of 64.4%. The homogenous preparation was obtained as shown by gel electrophoresis and ultracentrifugation. The antitumor activity of the enzyme both in vivo and in vitro is under investigation.
Assuntos
Aminoácido Oxirredutases/isolamento & purificação , Fungos Mitospóricos/enzimologia , Trichoderma/enzimologia , Cromatografia DEAE-Celulose , L-Aminoácido Oxidase , Especificidade por SubstratoRESUMO
A simple large-scale purification scheme for a trypsin-like enzyme from Actinomyces 771 was developed using carboxylic cation exchange resin Soloze K and DEAE-cellulose. The electrophoretically homogeneous enzyme with the specific trypsin activity of 1.4 U/mg was obtained with a yield of 53%.
Assuntos
Actinomyces/enzimologia , Fibrinolisina/isolamento & purificação , Tripsina/isolamento & purificação , Técnicas Bacteriológicas , Eletroforese em Gel de PoliacrilamidaRESUMO
Electrophoretically homogeneous proteolytic enzyme with molecular weight 31,500 and pI 3.75 was obtained from a culture medium of Streptomyces 771 by chromatography on N-benzyl chitin adsorbent, subsequent chromatography on CM-cellulose, and preparative isofocusing and chromatography on Sephadex G-75. The enzyme hydrolyzes N-benzoyl-DL-arginine-p-nitroanilide N-benzoyl-DL-lysine-p-nitro-anilide N-benzoyl-DL-arginine ethyl ester, and Na-caseinate. It also exhibits pronounced thrombolytic activity. The activity of the enzyme was suppressed by soya bean inhibitor, but remained unaffected by chelating agents and phenylmethylsulfonyl fluoride. The enzyme was immobilized on aldehyde dextran, and some kinetic parameters of the immobilized enzyme were determined. The thrombolytic activity of native and immobilized enzyme was studied as well.
Assuntos
Streptomyces/enzimologia , Tripsina/isolamento & purificação , Aminoácidos/análise , Proteínas de Bactérias/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Endopeptidases/farmacologia , Enzimas Imobilizadas , Metaloendopeptidases , Peptídeo Hidrolases , Inibidores da Tripsina/farmacologiaRESUMO
Chitin containing sorbents have been obtained for isolation and purification of serine proteases. Serine proteases from Bacillus subtilis have been purified 4-5 times and commercial preparations of trypsin and chymotrypsin 1.5-2 times by chromatography on nondeproteinized chitin. On the benzylated derivative of nondeproteinized chitin complete separation of trypsin and chymotrypsin has been achieved by chromatography of crude pancreatin. It has been shown that the protein moiety of chitin is important for preferential sorption of serine type proteases.
Assuntos
Bacillus subtilis/enzimologia , Endopeptidases/isolamento & purificação , Quitina , Cromatografia de Afinidade/métodos , Quimotripsina/isolamento & purificação , Serina Endopeptidases , Tripsina/isolamento & purificaçãoAssuntos
Peptídeo Hidrolases/isolamento & purificação , Animais , Bacillus subtilis/enzimologia , Ligação Competitiva , Cromatografia/métodos , Cromatografia de Afinidade , Quimotripsina/isolamento & purificação , Clostridium/enzimologia , Humanos , Concentração de Íons de Hidrogênio , Ligantes , Metaloproteínas , Colagenase Microbiana/isolamento & purificação , Concentração Osmolar , Papaína/isolamento & purificação , Pepsina A/isolamento & purificação , Tripsina/isolamento & purificaçãoRESUMO
Using biospecific adsorbent and subsequent gel-filtration of Sephadex G-75 three fractions of serine proteases (I--III) having different physicochemical properties were isolated from Bac. subtilis. The first protease had molecular weight of 23000--24000 (pH optimum 6,5, activation energy 16,6 ccal/mol. The second one had molecular weight of 29000, pH optimum 11,0, activation energy 14,4 ccal/mol. The third protease was a mixture of proteases with average molecular weights 26000 and pH optima at 7,0, 8,5 and 11,0.
Assuntos
Bacillus subtilis/enzimologia , Peptídeo Hidrolases/isolamento & purificação , Ativação Enzimática , Concentração de Íons de Hidrogênio , Substâncias Macromoleculares , Peso Molecular , Peptídeo Hidrolases/metabolismo , TermodinâmicaRESUMO
Metal and serine proteases were separated on the biospecific sorbent. Two different, homogeneous metal proteases were obtained by rechromatography of the metal protease. Activation energies, heat stability, molecular weights, influence of inhibitors, the dependence of activity on pH and temperature were determined. Properties of two metal proteases were compared with those of literary analogs.