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Eukaryotic Cell, April 2007, p. 674-681, Vol. 6, No. 4
1535-9778/07/$08.00+0     doi:10.1128/EC.00361-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Molecular and Biochemical Characterization of a Novel Intracellular Invertase from Aspergillus niger with Transfructosylating Activity{triangledown}

Coenie Goosen,1,2,{dagger} Xiao-Lian Yuan,3,{dagger} Jolanda M. van Munster,1,2 Arthur F. J. Ram,3 Marc J. E. C. van der Maarel,1,2,4* and Lubbert Dijkhuizen1,2

Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands,1 Centre for Carbohydrate Bioprocessing, TNO-University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands,2 Institute of Biology Leiden, Leiden University, Molecular Microbiology, Wassenaarseweg 64, 2333 AL, Leiden, The Netherlands,3 TNO Quality of Life, Business Unit Food and Biotechnology Innovations, Rouaanstraat 27, 9723 CC Groningen, The Netherlands4

Received 16 November 2006/ Accepted 30 January 2007

A novel subfamily 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 mass of 75 kDa, judging from sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Its optimum pH and temperature for sucrose hydrolysis were determined to be 5.0 and 37 to 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 a substrate, products up to a degree of polymerization 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 to 30% to total activity. At higher sucrose concentrations, FTF activity increased to up to 50% of total activity. Disruption of sucB in A. niger resulted in an earlier onset of sporulation on solid medium containing various carbon sources, whereas no alteration of growth in liquid culture medium was observed. SucB thus does not play an essential role in inulin or sucrose catabolism in A. niger but may be needed for the intracellular conversion of sucrose to fructose, glucose, and small oligosaccharides.

* Corresponding author. Mailing address: Centre for Carbohydrate Bioprocessing, TNO-University of Groningen, P.O. Box 14, 9750AA Haren, The Netherlands. Phone: 31 50 3632150. Fax: 31 50 3632154. E-mail: m.j.e.c.van.der.maarel{at}rug.nl

{triangledown} Published ahead of print on 9 February 2007.

{dagger} C.G. and X.-L.Y. contributed equally to this work.

Eukaryotic Cell, April 2007, p. 674-681, Vol. 6, No. 4
1535-9778/07/$08.00+0     doi:10.1128/EC.00361-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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