[Use of cadmium hydroxide gel for isolation of extracellular catalases from Penicillium piceum and characterization of purified enzymes].

We optimized the conditions for isolation of extracellular catalases from Penicillium piceum F-648 and P. piceum A3 by means of volume chromatography with cadmium hydroxide gel. Our study showed that 55-57 mg wet gel are sufficient for the maximum sorption of catalase from 1 ml of culture fluid. This gel was formed in 1 ml 70 mM Cd(NO3)2 after addition of NaOH (Cd(NO3)2/NaOH molar ratio 1:2.2). The eluting solution contained 50 mM NaH2PO4 (pH 7.0), 5.0 mM dithiothreitol, and 0.3% sodium cholate and was potent in desorbing catalase from the gel. Subsequent ultrafiltration of the eluate on the membrane with a retention limit of 50 kDa allowed us to concentrate and purify the sample from low-molecular-weight protein impurities. NH4Cl (1.0 M) containing 0.3% sodium cholate was used to wash the sample from low-molecular-weight aromatic metabolites. Purified catalases included 33-34% antiparallel beta-structures and 9% alpha-spirals. Under optimal conditions in the medium of 10 mM phosphate buffered saline (pH 7.0) at 30 degrees C, catalases from P. piceum F-648 were characterized by the following parameters: K(M), 158.8 mM; catalytic constant, 2.83 x 10(6) s(-1); enzyme inactivation rate constant in H2O2 decomposition, 3.5 x 10(-2) s(-1); and constant of the interaction between catalase complex I and second molecule of H2O2, 1.8 x 10(7) M(-1) s(-1).

[Experience in using a mobile digital fluorograph-equipped laboratory].

The authors share their experience in using a KFP-FC-RP digital fluorograph for mass fluorographic studies in a mobile fluorographic laboratory (on the basis of a ZIL-5301 EO automobile with a module body and an APCF-01 (ProScan-2000) fluograph made by ZAO "RENTGENPROM"). Studies were performed at the enterprises of Moscow and the Moscow Region. How the work of the mobile laboratory is organized is described. Since the able-bodied population was chiefly surveyed, the efficiency of the work can be considered rather high. The experience has shown that the use of a mobile fluorographic laboratory for mass examinations at the enterprises by a health care facility is much more effective and profitable than that of a permanent laboratory.

[Precipitated, coprecipitated, and carbon-mineral sorbents for isolation of extracellular catalase from Penicillium piceum F-648]. / Osazhdennye, soosazhdennye i uglemineral'nye adsorbenty dlia vydeleniia vnekletochnoi katalazy Penicillium piceum F-648.

The abilities of various sorbents to adsorb catalase (CAT; EC 1.11.1.6) from filtered culture liquid (FCL) of the fungus Penicillium piceum F-648 were compared. Potassium phosphate, hydroxyapatite (HAP), and coprecipitated sorbents containing calcium phosphate and magnesium hydroxide adsorbed extracellular CAT more efficiently than aluminum oxide, aluminum phosphate, or quartz sand. The enzyme was isolated from FCL of Penicillium piceum with the use of HAP and a binary coprecipitated sorbent, Ca3(PO4)2 + Mg(OH)2, 1:1 (CM). The CAT(CM) sample contained the least amount of protein admixture. Its spectra had absorption maximums at 279.6, 406.8 (Soret band), 540, 585, 636, and 703 nm and negative molar ellipticity minimums at 207 and 210-214 nm. The kinetic indices of the samples (KM, Vmax:KM, and specific activity) were intricately dependent on protein concentration in the reaction mixture. In dilute solutions, the KM and specific activities of CAT(CM) and CAT(HAP) equaled 667 and 137 mM; 300.9 x 10(4) and 30.0 x 10(4) U/mg protein, respectively. The effective velocity constants of inactivation of CAT(HAP), CAT(CM), and FCL in the reaction of H2O2 decomposition increased dramatically after dilution of samples. In the infinitely dilute solution, they were 4.30 x 10(-2), 6.46 x 10(-2), and 1.12 x 10(-2), respectively.

[Resistance of Penicillium piceum F-648 to hydrogen peroxide under short term and prolonged oxidative stress]. / Ustoichivost' Penicillium piceum F-648 k deistviiu peroksida vodoroda v usloviiakh kratkovremennogo i dlitel'nogo okislitel'nogo stressa.

Resistance of Penicillium piceum F-648 to hydrogen peroxide under short-term and prolonged oxidative stress was studied. An increase in the activity of intracellular catalase in fungal cells after short-term exposure to hydrogen peroxide was shown. Activation of fungal cells induced by H2O2 depends on H2O2 concentration, time of exposure, and the growth phase of the fungus. Variants of P. piceum F-648 that produced two forms of extracellular catalase with different catalytic properties were obtained due to prolonged adaptation to H2O2. Catalase with low affinity for substrate was produced predominantly by the parent culture and variant 3; however, a high substrate affinity of catalase was observed in variant 5. Variant 5 of P. peniceum F-648 displayed a high catalytic activity and operational stability of catalase in the presence of phosphate ions and the concentration of substrate less than 30 mM at pH more than 7.

[Kinetic characteristics of extracellular catalase from Penicillium piceum F-648 and variants of fungi, adapted to hydrogen peroxide]. / Kineticheskaia kharakteristika vnekletochnykh katalaz Penicillium piceum F-648 i variantov griba, adaptirovannykh k peroksidu vorodoroda.

A comparative kinetic study of extracellular catalases produced by Penicillium piceum F-648 and their variants adapted to H2O2 was performed in culture liquid filtrates. The specific activity of catalase, the maximum rate of catalase-induced H2O2 degradation (Vmax),Vmax/KM ratio, and the catalase inactivation rate constant in the enzymatic reaction (kin, s-1) were estimated in phosphate buffer (pH 7.4) at 30 degrees C. The effective constant representing the rate of catalase thermal inactivation (kin*, s-1) was determined at 45 degrees C. In all samples, the specific activity and KM for catalase were maximum at a protein concentration in culture liquid filtrates of 2.5-3.5 x 10(-4) mg/ml. The effective constants describing the rate of H2O2 degradation (k, s-1) were similar to that observed in the initial culture. These values reflected a twofold decrease in catalase activity in culture liquid filtrates. We hypothesized that culture liquid filtrates contain two isoforms of extracellular catalase characterized by different activities and affinities for H2O2. Catalases from variants 5 and 3 with high and low affinities for H2O2, respectively, had a greater operational stability than the enzyme from the initial culture. The method of adaptive selection for H2O2 can be used to obtain fungal variants producing extracellular catalases with improved properties.

[Preparation and characteristics of mutant strains of Penicillium funiculosum--producers of glucose oxidase]. / Poluchenie i kharakteristika mutantnykh shtammov Penicillium funiculosum--produtsentov gliukozooksidazy.

After the mutagenesis of Penicillium funiculosum with UV light and N-nitroso-N-methylurea, 83 of 2237 grown colonies were surrounded with increased zones of glucose oxidase diffusion. Analysis of the glucose oxidase activity of selected mutant strains grown in submerged cultures allowed 18 mutant strains to be obtained whose glucose oxidase activity was 5-153% higher (in a medium with glucose) and 4-83% higher (in a medium with sucrose) than that of the parent strain. Two of these mutant strains, UV6.31 and NMU95-132, possessed high glucose oxidase activity when grown in media with glucose or sucrose and produced large amounts of mycelia. The active and morphologically stable mutant P. funiculosum NMU95-132 was chosen for further selection work.