Molecular and biochemical characterization of a novel intracellular invertase from Aspergillus niger with transfructosylating activity.
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 degrees 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 K(m), K(i), and V(max) of 2.0 +/- 0.2 mM, 268.1 +/- 18.1 mM, and 6.6 +/- 0.2 mumol 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.
Goosen
Coenie
C
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands.
Yuan
Xiao-Lian
XL
van Munster
Jolanda M
JM
Ram
Arthur F J
AF
van der Maarel
Marc J E C
MJ
Dijkhuizen
Lubbert
L
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000187:drug effects / Q000201:enzymology*; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D005632:Fructose / Q000378:metabolism*; D017353:Gene Deletion; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010802:Phylogeny; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity / Q000187:drug effects; D013395:Sucrose / Q000494:pharmacology; D013696:Temperature; D043324:beta-Fructofuranosidase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D005632:Fructose; D013395:Sucrose; D043324:beta-Fructofuranosidase