CHARACTERIZATION OF ACID INVERTASE FROM THE SNOW MOLD MONOGRAPHELLA-NIVALIS - A MESOPHILIC ENZYME FROM A PSYCHROPHILIC FUNGUS

A soluble acid invertase was extracted by mechanical disruption of myc elium of the psychrophile Monographella nivalis grown on sucrose in su bmerged culture. The crude preparation was stable to incubation at pH 6.2 for 1 h at temperatures up to 47 degrees C and was stable to handl ing at room temperature. Half of the initial activity was lost after 1 h at 52 degrees C and all activity was lost after 1 h at 57 degrees 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 alpha-methyl mannopyranosidase indicating the enzyme to be a mannose-containing glycoprotein. By gel filtration, th e apparent M(r) was determined at 195 kDa. The invertase was purified 106-fold by salt precipitation. The partially purified enzyme exhibite d maximal activity at pH 4.2 and apparent Michaelis constants for sucr ose of 1.2, 2.0 and 2.6 mM at 3, 9 and 15 degrees C. The activity incr eased exponentially with temperature in the range 7-55 degrees C. Q(10 ) fell with increased temperature giving values of 1.96 between 5 and 15 degrees C and 1.60 between 40 and 50 degrees C. Maximal activity wa s recorded at 55 degrees C. Arrhenius analysis of temperature data in the range 7-52 degrees C produced a continuous linear relationship. Th e activation energy for sucrose hydrolysis was 38.8 kJ mol(-1). The th ermal stability and thermal kinetic properties of the invertase were s imilar to those of invertases from mesophilic organisms. The invertase catalysed fructosyl transfer at 13% of the molar activity against suc rose when assayed under conditions analogous to those in culture. The major fructan products were neokestose, isokestose, kestose and an uni dentified tetrasaccharide. Traces of larger fructans were also detecte d. The transient accumulation of fructan in cultures of M. nivalis can be explained as a side reaction of invertase activity. The enzymologi cal and physiological data do not suggest a cryoprotective function fo r fructan during the growth of the fungus.