Rt. Kushi et al., Production, purification and characterization of an extracellular inulinase from Kluyveromyces marxianus var. bulgaricus, J IND MIC B, 25(2), 2000, pp. 63-69
Citations number
32
Categorie Soggetti
Biotecnology & Applied Microbiology",Microbiology
Journal title
JOURNAL OF INDUSTRIAL MICROBIOLOGY & BIOTECHNOLOGY
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.