Production, purification and characterization of an extracellular inulinase from Kluyveromyces marxianus var. bulgaricus

The yeast Kluyveromyces marxianus var. bulgaricus produced large amounts of extracellular inulinase activity when grown on inulin, sucrose, fructose a nd glucose as carbon source, This protein has been purified to homogeneity by using successive DEAE-Trisacryl Plus and Superose 6 HR 10/30 columns. Th e purified enzyme showed a relative molecular weight of 57 kDa by sodium do decyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and 77 kDa by g el filtration in Superose 6 HR 10/30, Analysis by SDS-PAGE showed a unique polypeptide band with Coomassie Blue stain and nondenaturing PAGE of the pu rified enzyme obtained from media with different carbon sources showed the band, too, when stained for glucose oxidase activity, The optimal hydrolysi s temperature for sucrose, raffinose and inulin was 55 degrees C and the op timal pH for sucrose was 4.75, The apparent K-m values for sucrose, raffino se and inulin are 4.58, 7.41 and 86.9 mg/ml, respectively, Thin layer chrom atography showed that inulinase from K. marxianus var. bulgaricus was capab le of hydrolyzing different substrates (sucrose, raffinose and inulin), rel easing monosaccharides and oligosaccharides, The results obtained suggest t he hypothesis that enzyme production was constitutive.