Secretion of two beta-fructofuranosidases by Aspergillus niger growing in sucrose.
Aspergillus niger is induced to secrete two invertases, named SUC 1 and SUC 2, when grown on a minimal medium containing sucrose. Although, both have been classified as beta-D-fructofuranoside fructohydrolases, SUC 2 also possesses inulin hydrolytic activity (sucrose/inulin activity ratio of 4). These activities have been separated from each other and almost completely purified by anion-exchange, lectin affinity chromatography, and chromatofocusing. SUC 1 appeared as a single glycoprotein band on PAGE and SDS-PAGE corresponding in size to 250 and 125 kDa, respectively, compared with a much broader band (suggesting greater glycan heterogeneity) of 210-240 and 90-120 kDa for SUC 2. Therefore, both may be dimers, in their natural conformation. The glycan part of both contained the same monosaccharides: mannose, glucose, galactose, and N-acetylglucosamine; however, SUC 1 had approximately 10-fold more mannose and this was utilized to separate it from SUC 2 by Galanthus nivalis lectin affinity. Both the apparent Km values and the pH activity curves were different; SUC 1 did not show normal Michaelis-Menten kinetics to sucrose and apparent Michaelis constants of 30 and 160 mM were obtained. Activity was observed over a large range of pH 4.5-9 with a maximum at pH 6. In contrast, SUC 2 exhibited a Km of 40 and 1.7 mM to sucrose and inulin, respectively, with a pH optimum of 5.0 for both. Treatment with endo-beta-N-acetylglucosaminidase suggests that in both SUC 1 and SUC 2 some of the glycan present was N-linked glycan but that the differences in enzyme activities were not due to the N-linked moiety.
Wallis
G L
GL
Department of Biochemistry, Medical School, University of Nottingham, United Kingdom.
Hemming
F W
FW
Peberdy
J F
JF
D001234:Aspergillus niger / Q000201:enzymology* / Q000254:growth & development; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction; D027801:Galanthus; D006026:Glycoside Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D013395:Sucrose / Q000378:metabolism*; D043324:beta-Fructofuranosidase
D007527:Isoenzymes; D013395:Sucrose; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase