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A soluble acid invertase was extracted by mechanical disruption of mycelium of the psychrophile Monographella nivalis grown on sucrose in submerged culture. The crude preparation was stable to incubation at pH 6.2 for 1 h at temperatures up to 47 °C and was stable to handling at room temperature. Half of the initial activity was lost after 1 h at 52 °C and all activity was lost after 1 h at 57 °C. A single isoform with activity against sucrose was detected on both native PAGE and IEF activity gels, exhibiting an isoelectric point of pH 3.6. The activity bound tightly to Concanavalin A-sepharose and was not displaced by 500 mM α-methyl mannopyranosidase indicating the enzyme to be a mannose-containing glycoprotein. By gel filtration, the apparent Mr was determined at 195 kDa. The invertase was purified 106-fold by salt precipitation. The partially purified enzyme exhibited maximal activity at pH 4.2 and apparent Michaelis constants for sucrose of 1.2, 2.0 and 2.6 mM at 3, 9 and 15 °C. The activity increased exponentially with temperature in the range 7-55 °C. Q10 fell with increased temperature giving values of 1.96 between 5 and 15 °C and 1.60 between 40 and 50°C. Maximal activity was recorded at 55 °C. Arrhenius analysis of temperature data in the range 7-52 °C produced a continuous linear relationship. The activation energy for sucrose hydrolysis was 38.8 kJ mol-1. The thermal stability and thermal kinetic properties of the invertase were similar to those of invertases from mesophilic organisms. The invertase catalysed fructosyl transfer at 13% of the molar activity against sucrose when assayed under conditions analogous to those in culture. The major fructan products were neokestose, isokestose, kestose and an unidentified tetrasaccharide. Traces of larger fructans were also detected. The transient accumulation of fructan in cultures of M. nivalis can be explained as a side reaction of invertase activity. The enzymological and physiological data do not suggest a cryoprotective function for fructan during the growth of the fungus. [ABSTRACT FROM AUTHOR]

An asporogenous strain of the pink snow mould fungus, Monographella nivalis (Schaffnit) E. Müller, anamorph Gerlachia nivalis Ces. ex Sac.) W. Gams & E. Müller (Syn. Fusarium nivale Ces. ex Sacc.), grew at 5 °C on a defined salts medium plus vitamins and utilized a variety of simple and polymeric carbohydrates as the sole carbon and energy source. Mycelium was grown at temperatures between 3 and 15 °C in aerated submerged fermentation culture in chemically defined mediam plus sucrose. Optimum growth rates of 0.035-0.033 h-1 occurred between 9 and 12 °C. Growth in a simple medium showed that all biochemical and physiological processes necessary for growth were functional at 3 °C. The growth performance of the organism at low temperatures was no better than would be expected from extrapolation of mesophilic growth responses to temperature. The optimum growth temperature of 9-12 °C showed that some biochemical or physiological process was impaired above 12 °C. Uptake and incorporation of 35S-methionine by mycelium at different temperatures showed that general protein synthesis increased up to 25 °C, and hence was not responsible per se for the sensitivity to temperatures above 12 °C. Heat shock proteins were synthesized at the relatively low temperature of 25 °C, consistent with the low temperature optimum for growth. When grown with sucrose as the sole carbon source, the mycelium catalyzed the extracellular hydrolysis of sucrose, releasing glucose and fructose together with a small amount of fructan trisaccharides and a trace of tetra- and penta-saccharides. Fructan accumulation was transient, corresponding with maximal rates of sucrose hydrolysis. Most biomass formation occurred in the absence of fructan in the culture, hence fructan was not necessary for growth at low temperature and did not appear to function as a cryoprotectant. Invertase activity was mostly (60-70%) bound to mycelium; the remainder was free in the culture supernatant. The regulation of invertase expression appeared to be by sucrose-induction, rather than by end-product repression. Rates of sucrose hydrolysis in culture were temperature-sensitive and were markedly depressed above 12c, indicating inhibition of invertase formation. [ABSTRACT FROM AUTHOR]