The properties of the extracellular alkaline phosphatase of the causative agent of melioidosis]

After growing P. pseudomallei VPA on solid medium extracellular alkaline phosphatase with a molecular weight of 93,000 AMU was isolated, and practically purified from the extract of this medium by precipitation with ammonium sulfate, subsequent gel chromatography and concentration on membrane filters. The optimum conditions for enzymatic reaction were found to be pH 9.0 and a temperature of 50 degrees C. The enzyme was resistant to freezing and to heating at a temperature of up 60 degrees C for 30 minutes, as well as to the action of pH 3.0-10.5, but became completely inactivated after heating at 90 degrees C for 10 minutes and incubation at pH 2.0 for 20 hours.     

Kinetic properties of extracellular alkaline protease of Rhizopus oryzae

Production of alkaline protease by Rhizopus species is an unusual phenomenon. However, a locally isolated species of Rhizopus oryzae was found to secrete alkaline serine protease of industrial importance. The kinetic parameter V (reaction velocity) of the purified fractionated enzyme was evaluated under different environmental conditions and substrate to enzyme ratios. The Michaelis constant (K_m) and maximum reaction velocity (V_max) were also estimated.     

Kinetic properties of chitinase-1 from the fungal pathogen Coccidioides immitis

The endochitinase from Coccidioides immitis (CiX1) is a member of the class 18 chitinase family. Here we show the enzyme functions by a retaining catalytic mechanism; that is, the beta-conformation of the chitin substrate linkages is preserved after hydrolysis. The pattern of cleavage of N-acetyglucosamine (GlcNAc) oligosaccharide substrates has been determined. (GlcNAc)6 is predominantly cleaved into (GlcNAc)2 and (GlcNAc)4, where the (GlcNAc)2 group arises from the nonreducing end of the substrate and is formed as the beta-anomer. With time, transglycosylation occurs, generating (GlcNAc)8 from the product dimer and fresh hexamer. Similar patterns are seen for the cleavage of (GlcNAc)5 and (GlcNAc)4 where dimers cleaved from the nonreducing end reflect the most common binding and hydrolysis pattern. Intrinsic fluorescence measurements suggest the dissociation constant for (GlcNAc)4 is 50 microM. Synthetic substrates with fluorescent leaving groups exhibit complicated profiles in the relationship between initial velocity and substrate concentration, making it difficult to obtain the values of kinetic constants. An improved theoretical analysis of the time-course of (GlcNAc)6 degradation allows the unitary free energy of binding of the individual subsites of the enzyme to be estimated. The free energy values obtained are consistent with the dissociation constant obtained by fluorescence measurements, and generate a model of substrate interaction that can be tested against the crystal structure of the enzyme.     

Purification and properties of extracellular xylan hydrolases ofStreptomyces exfoliatus

Fractionation of extracellular xylan hydrolases of a strain ofStreptomyces exfoliatus MC₁ (by salting out, molecular sieving and ion exchange chromatography) revealed the presence of five species of the enzyme. Three major fractions could be purified to homogeneity; two were apparently endohydrolases and the third an exo-xylan hydrolase. The three fractions showed different degrees of affinity to the substrate and differed considerably in their substrate specificities. One of the endo-enzymes was specific to xylan while the other could also attack cellulose, inulin and pectin. The exo-enzyme showed xylanolytic and cellulolytic functions only. The three fractions further differed in their response to the presence of metal ions, mercapto reagents and compounds. Although the pH and temperature optima were different, the three fractions functioned synergistically in the hydrolysis of xylan.     

Serological Comparison of Spherules and Arthrospores of Coccidioides immitis

Spherule and arthrospore cellular preparations were sonic-treated and separated into their respective supernatant and sediment components. Complement-fixation tests with antispherule and antiarthrospore pooled rabbit sera revealed that the soluble antigens exhibited more serological activity than the sediment preparations. After autoclaving, an arthrospore cellular antigen exhibited increased activity with either antisera, whereas autoclaved spherules exhibited increased activity only with antispherule serum. Complement-fixation tests with coccicioidin and spherule culture supernatant preparations revealed quantitative or qualitative differences in antigenic determinants between these two morphological phases of Coccidioides immitis.     

Screening Brazilian Macrophomina phaseolina isolates for alkaline lipases and other extracellular hydrolases

Macrophomina phaseolina, phylum Ascomycota, is a phytopathogenic fungus distributed worldwide in hot dry areas. There are few studies on its secreted lipases and none on its colony radial growth rate, an indicator of fungal ability to use nutrients for growth, on media other than potato-dextrose agar. In this study, 13 M. phaseolina isolates collected in different Brazilian regions were screened for fast-growth and the production of hydrolases of industrial interest, especially alkaline lipases. Hydrolase detection and growth rate determination were done on citric pectin, gelatin, casein, soluble starch, and olive oil as substrates. Ten isolates were found to be active on all substrates tested. The most commonly detected enzymes were pectinases, amylases, and lipases. The growth rate on pectin was significantly higher (P < 0.05), while the growth rates on the different media identified CMM 2105, CMM 1091, and PEL as the fastest-growing isolates. The lipase activity of four isolates grown on olive oil was followed for 4 days by measuring the activity in the cultivation broth. The specific lipolytic activity of isolate PEL was significantly higher at 96 h (130 mU mg protein-1). The broth was active at 37 °C, pH 8, indicating the potential utility of the lipases of this isolate in mild alkaline detergents. There was a strong and positive correlation (0.86) between radial growth rate and specific lipolytic activity.     

Isolation and identification of Coccidioides immitis from natural sources

The review of media and techniques that have been developed to date appears to provide more than adequate choice for investigators in endemic areas to perform ecological studies of this organism. The final identification of this organism still lies in the demonstration of itsin vivo morphology.Paper read at the Eighth International Congresses for Tropical Medicine and Malaria, September 1968, Teheran (Iran).     

Morphogenesis throughout saprobic and parasitic cycles of Coccidioides immitis

The fungus, Coccidioides immitis, differs from other dimorphic pathogens in that its parasitic stage is a complex morphogenic cycle, raising the question that changes in structure and composition during morphogenesis might influence host responses. As a prelude to examining the interaction of fungal morphogenesis and host responses, the life cycle of this fungus has been examined in greater detail than previously accomplished. During saprobic development, alternating enterothallic arthroconidia are formed as infectious propagules. The outer wall is broken and loosely adherent. Under in vitro conditions supporting the parasitic cycle, multinucleate arthroconidia transform into uninucleate round cells. Rapid, synchronous, nuclear replication is initiated, accompanied by increase in cell mass and deposition of new cell wall substance. As karyokinesis ceases, morphologic differentiation begins with invagination of the inner layers of the spherule wall and then is progressive, eventually segmenting the protoplasm into uninucleate endospores grouped in clusters within a hyaline membrane. Endospores, escaping through a break in the spherule wall, are held in aggregates by fibrils which are stretched and broken as endospores separate. It would seem that rapid production of hundreds of progeny from an original single cell, protected during development by an enclosing spherule wall and then released in clusters, should favor establishment of the fungus in a host, and dynamic changes in the cell wall during morphogenesis should influence the host response.     

Dimorphism in the fungus Coccidioides immitis Rixford et Gilchrist under the action of saprophytic bacilli]

It is shown that there are specific substances produced and secreted into the environment by saprotrophic bacilli. These inhibit the growth of the coccidioidal fungus in its mycelial form and some cells are converted into the yeast form, which leads to the destruction of the fungi (in natural environment) or, if the conditions allow, to their growth in the yeast form. This phenomenon, existence of a large amount of bacilli antagonistic to Coccidioides immitis, may be one of reasons why the latter has not been isolated so far from the soil in the territory of the USSR.     

Kinetic properties of de-repressible alkaline phosphatase (EC 3.1.3.1) from Neurospora crassa

De-repressible alkaline phosphatase from N. crassa shows inhibition by PNP-P and a hyperbolic mixed-type inhibition by Pi. Both increasing concentrations of Pi and decreases in assay pH abolished inhibition by the substrate. Also, Pi promoted polymerization of the enzyme molecule, whose effect may account for the inhibitory behaviour shown by the enzyme in the presence of low Pi concentrations.