Secretion of Two β-Fructofuranosidases byAspergillus nigerGrowing in Sucrose

• Published in: Archives of biochemistry and biophysics 1997-09, Vol.345 (2), p.214-222
• Main Authors: Wallis, G.L.F., Hemming, F.W., Peberdy, J.F.
• Format: Article
• Language: English
• Subjects: A. niger; extracellular enzyme; β-Fructofuranosidase (invertase, inulinase)
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1006/abbi.1997.0228

Aspergillus nigeris 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 β-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, andN-acetylglucosamine; however, SUC 1 had approximately 10-fold more mannose and this was utilized to separate it from SUC 2 byGalanthus nivalislectin affinity. Both the apparentKmvalues 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 mmwere obtained. Activity was observed over a large range of pH 4.5–9 with a maximum at pH 6. In contrast, SUC 2 exhibited aKmof 40 and 1.7 mmto sucrose and inulin, respectively, with a pH optimum of 5.0 for both. Treatment with endo-β-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.