Molecular and Biochemical Characterization of a Novel Intracellular Invertase fromAspergillus nigerwith Transfructosylating Activity

Abstract

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 MichaelisMenten 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 mol 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.Fructans and fructooligosaccharides (FOS) consist of a chain of fructose molecules linked to a terminal glucose residue. These fructose monomers are linked by either ␤2,1 (inulin) or ␤2,6 (levan) glycosidic bonds. Inulin and levan have several favorable properties that make them commercially interesting for applications in pharmaceutical and food industries (36). In the human digestive tract, FOS are almost exclusively fermented by bifidobacteria and lactobacilli, which have beneficial health effects (20,31,33). Commercially, FOS are produced by the enzymatic hydrolysis of inulin isolated from plants, primarily chicory and Jerusalem artichoke (36). Alternatively, sucrose can be converted into FOS by using a range of different transfructosylating enzymes originating from plants, bacteria, and fungi (36

Plant-Derived Sucrose Is a Key Element in the Symbiotic Association between Trichoderma virens and Maize Plants

“…Protein extracts were prepared, and the enzyme was purified through anion-exchange chromatography followed by gel-filtration chromatography (data not shown). The purified enzyme from T. virens displayed biochemical properties similar to those described for the intracellular Inv from A. niger (Goosen et al, 2007;Fig. 3).…”

“…S1). The deduced amino acid sequence is 40% identical to the S. cerevisiae Inv and similar to SucB, the intracellular acid Inv from A. niger (Goosen et al, 2007). TvInv does not contain a transit peptide for secretion or glycosylation sites.…”

“…TvInv is an intracellular Inv with an acidic optimum pH for Suc hydrolysis, and the enzyme displays b-fructofuranosidase activity with biochemical properties similar to those described for an intracellular Inv (SacB) recently characterized from A. niger ( Fig. 3; Goosen et al, 2007). BLAST searches in the T. virens genome sequence, using S. cerevisiae Ac-Inv and SacB as queries, retrieved only one homologous sequence (tvinv) displaying the eight motifs conserved in characterized b-fructofuranosidases ( Fig.…”

“…1A). The deduced amino acid sequence of the putative protein displayed the eight conserved motifs associated with catalytic activity in known Inv with acidic optimum pH (Goosen et al, 2007;Supplemental Fig. S1).…”

Cloning, Expression and Characterization of a Novel Fructosyltransferase from Aspergillus oryzae ZZ-01 for the Synthesis of Sucrose 6-Acetate

“…The majority of the ftase genes reported to date have been isolated from Aspergillus strains of fungi, although a recent database mining analysis identified ftase-related sequences in the genomes of Gibberella zeae, Magna porthe grisea and Ustilago maydis [19]. The Aspergillus genes become increasingly homogeneous at sizes in the range of 1.6-2.2 kb and encode for enzymes of about 600 amino acids in length [8,[19][20][21]. In bacteria, the ftase genes vary in size from 1.2 kb in Thermotoga maritima [22] up to 4.4 kb in Leuconostoc citreum [23], producing enzymes containing 432 and 1466 amino acids, respectively.…”