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 he 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.
Arrhenius activation energy
- Con A
Optimum temperature for catalysis
Triphenyl tetrazolium chloride
Threshold for thermal inactivation