Peter Rapp

Formation, Isolation and Characterization of Trehalose Dimycolates from Rhodococcus erythropolis Grown on n-Alkanes

Summary: Rhodococcus erythropolis DSM 43215 produced a surface-active trehalose lipid whose formation was induced by n-alkanes to a maximum of 2.1 g l-1 in a 50 l batch culture on 2% (w/v) n-alkanes of chain length C12 to C18. The glycolipid was extracted from the biomass with n-hexane and was purified by repeated chromatography on silica gel. It contained α,α-trehalose as the sole non-reducing sugar. The lipid moiety was characterized by 13C nuclear magnetic resonance spectroscopy and mass spectrometry and consisted predominantly of saturated long-chain α-branched β-hydroxy fatty acids (mycolic acids) ranging from C32H64O3 to C38H76O3, of which C34H68O3 and C35H70O3 predominated. The molar ratio of trehalose to mycolic acids was 1:2. 13C nuclear magnetic resonance analysis of the O-hexamethyltrehalose obtained by saponification of the permethylated trehalose dimycolates revealed, with the aid of deuterium exchange, that the ester linkages of mycolic acids are to both primary alcohol groups at the C-6 and C-6’ positions of the trehalose.

1,3-β-Glucanase, 1,6-β-GIucanase and β-Glucosidase Activities of Sclerotium glucanicum: Synthesis and Properties

Summary: The filamentous fungus Sclerotium glucanicum excreted significant amounts of 1,3-β-glucanase, 1,6-β-glucanase and β-glucosidase activities when the culture medium was depleted of carbon sources. During starvation small amounts of intracellular 1,3-β- and 1,6-β-glucanase and β-glucosidase activities were also detected. Very low levels of β-glucanase activity remained bound to mycelium and some activity was found loosely attached to the cells and/or to water-soluble 1,3-β-/1,6-β-glucan adhering to the cell walls. During active growth intracellular 1,3-β-glucanase and mycelium-bound 1,3-β- and 1,6-β-glucanase activities were detected in small or trace amounts. During hyphal growth very low levels of 1,3-β- and 1,6-β-glucanase activities were also found to be weakly associated with the cells and/or with water-soluble β-glucan covering the hyphae. Cycloheximide inhibited the increase in intra- and extracellular 1,3-β- and 1,6-β-glucanase and β-glucosidase activities. This indicated that de novo protein synthesis was involved in the intra- and extracellular appearance of these three enzyme activities in derepressed cells. The formation of the extracellular 1,3-β-glucanase, 1,6-β-glucanase and β-glucosidase activities was regulated by catabolite repression. 1,3-β- and 1,6-β-glucanase activities were uncompetitively inhibited and β-glucosidase activity noncompetitively inhibited by glucose and glucono-δ-lactone. Optimum pH and temperature values as well as thermal stabilities of the three extracellular enzyme activities were determined. Almost all of the β-glucosidase activity but only one-third of the extracellular 1,3-β- and 1,6-β-glucanase activities were found to bind to Con A-Sepharose. Under conditions of carbon limitation almost 90% of the extracellular 1,3-β-/1,6-β-glucan excreted during fungal growth was degraded by the extracellular β-glucanases.

Multiphasic Kinetics of Transformation of 1,2,4-Trichlorobenzene at Nano- and Micromolar Concentrations by Burkholderia sp. Strain PS14

ABSTRACT The transformation of 1,2,4-trichlorobenzene (1,2,4-TCB) at initial concentrations in nano- and micromolar ranges was studied in batch experiments with Burkholderia sp. strain PS14. 1,2,4-TCB was metabolized from nano- and micromolar concentrations to below its detection limit of 0.5 nM. At low initial 1,2,4-TCB concentrations, a first-order relationship between specific transformation rate and substrate concentration was observed with a specific affinity (a0A) of 0.32 liter · mg (dry weight)−1 · h−1 followed by a second one at higher concentrations with anaoA of 0.77 liter · mg (dry weight)−1 · h−1. This transition from the first-order kinetics at low initial 1,2,4-TCB concentrations to the second first-order kinetics at higher 1,2,4-TCB concentrations was shifted towards higher initial 1,2,4-TCB concentrations with increasing cell mass. At high initial concentrations of 1,2,4-TCB, a maximal transformation rate of approximately 37 nmol · min−1 · mg (dry weight)−1 was measured, irrespective of the cell concentration.

1,3-β-Glucan synthase in cell-free extracts from mycelium and protoplasts of Sclerotium glucanicum

Conditions for obtaining stable protoplasts from Sclerotium glucanicum and their reversion to hyphal growth were determined. 1,3-beta-Glucan synthase activity was detected in particulate enzyme fractions from mycelium and protoplasts of Scl. glucanicum. UDP-[U-14C]glucose was linearly incorporated into a beta-glucan for about 20 min at 25 degrees C. Optimum pH and temperature values, as well as thermal stabilities of the 1,3-beta-glucan synthase activity, were determined. High concentrations of EDTA were inhibitory. Enzyme activity was stimulated by ATP and GTP. The apparent Km value for UDP-glucose was 0.54 mM. The reaction product was characterized as 1,3-beta-glucan by 13C NMR spectroscopy and hydrolysis products of an exo-1,3-beta-glucanase.

1,4–3-GLUCANASES AND 1,4-β-GLUCOSIDASES FROM PENICILLIUM JANTHINELLUM: PRODUCTION, LOCATION, ADSORPTION AND REGULATION

ABSTRACT Production of 1,4-β-glucanases and 1,4-β-glucosidases and their location have been investigated in cultures of Penicillium janthinellum , grown on cellulose, and mono- and disaccharides. 1,4-β-Glucosidases were formed constitutively and found to be predominantly cell free. Only a small amount was cell bound throughout the cultivations. The endo-1,4-β-glucanases, on the other hand, were excreted into the medium. Their formation was induced by cellobiose and controlled by catabolite repression. Both 1,4-β-glucanase activities, towards Avicel and towards CM-cellulose, were inhibited competitively by glucose and cellobiose. For the determination of the total endo-1,4-β-glucanase activity of culture broths, containing solids and mycelia, a new rotational viscosimetric method was used. This method is based upon the very rapid and complete desorption of the endo-1,4-β-glucanases from insoluble cellulose by an excess of Na-CM-cellulose and upon the viscosity lowering effect of the endo-1,4-β-glucanases on the Na-CM-cellulose solution. By this method the endoglucanase activity could be measured not only in the culture filtrate but also when located at the cell wall or adsorbed on residual Avicel. The degree of adsorption of endo-1,4-β-glucanases by residual Avicel during batch cultivations was proportional to the decreasing Avicel concentration in the culture broth. The adsorption of soluble protein and endo-1,4-β-glucanases by Avicel was dependent on temperature but independent of the pH-value within the range 6.0 to 3.0. KEYWORDS Penicillium janthinellum; 1,4-β-glucanases; 1,4-β-glucosidases; enzyme production; adsorption; endoglucanase activity determination; induction; catabolite repression; inhibition.