Eukaryotic Cell doi:10.1128/EC.00361-06
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
Molecular and Biochemical Characterization of a Novel Intracellular Invertase from Aspergillus niger with Transfructosylating Activity
Coenie Goosen,
Xiao-Lian Yuan,
Jolanda M van Munster,
Arthur F.J. Ram,
Marc J.E.C. van der Maarel*,
and
Lubbert Dijkhuizen
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands; Centre for Carbohydrate Bioprocessing TNO-University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands; Institute of Biology Leiden, Leiden University, Molecular Microbiology, Wassenaarseweg 64, 2333 AL, Leiden, The Netherlands; TNO Quality of Life, Business Unit Food and Biotechnology Innovations, Rouaanstraat 27, 9723 CC Groningen, The Netherlands
* To whom correspondence should be addressed. Email:
m.j.e.c.van.der.maarel{at}rug.nl.
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Abstract |
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A novel sub-family of putative intracellular invertase enzymes (glycoside hydrolase family 32) has previously been identified in fungal genomes. Here we report phylogenetic, molecular and biochemical characteristics of SucB, one of two novel intracellular invertases identified in Aspergillus niger. The sucB gene was expressed in Escherichia coli and an invertase negative strain of Saccharomyces cerevisiae. Enzyme purified from E. coli lysate displayed a molecular weight of 75 kDa, judging from SDS-PAGE analysis. Its optimum pH and temperature for sucrose hydrolysis were determined to be 5.0 and 37-40°C, respectively. In addition to sucrose, the enzyme hydrolyzed 1-kestose, nystose and raffinose, but not inulin and levan. SucB produced 1-kestose and nystose from sucrose and 1-kestose, respectively. With nystose as substrate, products up to a degree of polymerization (DP) of 4 were observed. SucB displayed typical Michaelis-Menten kinetics with substrate inhibition on sucrose (apparent Km, Ki, and Vmax of 2.0 (±0.2) mM, 268.1 (±18.1) mM and 6.6 (±0.2) µmol min-1 mg-1 of protein (total activity) respectively). At sucrose concentrations up to 400 mM, transfructosylation (FTF) activity contributed approximately 20-30% to total activity. At higher sucrose concentrations, FTF activity increased up to 50% of total activity. Disruption of sucB in A. niger resulted in earlier onset of sporulation on solid media containing various carbon sources, whereas no alteration of growth in liquid culture media was observed. SucB thus does not play an essential role in inulin or sucrose catabolism in A. niger, but may be needed for intracellular conversion of sucrose to fructose, glucose and small oligosaccharides.
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