Identification and characterization of glycoside hydrolase family 32 enzymes from Aspergillus niger

This thesis focuses on the identification and characterization of sucrose and fructan modifying enzymes present in the genome of the industrially important filamentous fungus, Aspergillus niger. In addition to three known activities, encoded by the genes suc1 (invertase activity; designated sucA), inuE (exo-inulinase activity) and inuA/inuB (endo-inulinase activity), two new putative intracellular invertase-like proteins were identified (designated sucB and sucC; Chapter 2, Yuan et al., 2006; Pel et al., 2007). These enzymes were similarly regulated and all strongly induced on sucrose and inulin. sucB, but not sucC, is expressed at a low level, and up-regulated in sucrose- or inulin-containing media. Biochemical analysis of heterologously expressed SucB indicated that it is an invertase with high substrate affinity for sucrose (Chapter 3, Goosen et al., 2007). Disruption of the sucB gene in A.niger indicated that it was not essential for sucrose or fructan metabolism; however, it might be (in)directly responsible for the observed early onset of sporulation. The presence of SucB homologues in other fungal species indicates that these proteins may all play an important, but not essential role in the intracellular metabolism of sucrose (Chapters 2 and 3; Yuan et al., 2006; Goosen et al., 2007). Heterologous expression, purification and characterization of the extracellular exo-inulinase AngInuE (InuE) showed that his monomeric enzyme completely hydrolyses inulin, levan and sucrose to free fructose and glucose. AngInuE also produces small amounts of inulin-, levan and neo-series inulo-oligosaccharides from sucrose, 1-kestose and nystose (Chapter 4, Goosen et al., submitted for publication). Finally, site-directed mutagenesis in the putative inulin-binding domain (Ser469, domain G) of AngInuE indicated that this domain was essential for activity of this enzyme (Chapter 4, Goosen et al., submitted for publication)