Aspergillusis a filamentous fungus that can grow in many environments, on several substrates at different conditions. In the soil,Aspergillirecycle nutrients by the degradation of plant material. In particular,Aspergilliare known for their capability to spoil plant materials like grains and fruits. In the industryAspergilliare often being used as hosts for the production of both homologous andheterologous(foodgrade) enzymes.In the food industry aminopeptidases from A.nigerare being used for the production of cheese, for baking and the preparation of soy hydrolysates. The use ofAspergillias hosts for the production of industrial proteins has resulted in detailed studies on theproteolyticspectra. Although theendopeptidasespectrum is thoroughly studied, the aminopeptidase spectrum is not. In this thesis aminopeptidases of A.nigerare analyzed. Aminopeptidases are classified according to their catalytic activity intometallo, serine andcysteineaminopeptidases.AminopeptidaseA (ApsA) was cloned using conserved regions within the M1 family ofmetalloaminopeptidases. The derived amino acid sequence of this aminopeptidase is highly similar to two yeast aminopeptidases. The protein wasoverexpressedin A.nigerand the purified product was characterized.ApsAprefers lysine at the N-terminal end of a peptide. The K M andK catfor the artificial substrate K-pNAis 0.17mMand0.49mkatmg -1. The pH optimum of the enzyme is between pH 7.5 and 8.A conserved region ofApsAwas used to search for aparalog. Indeed aparalogwas found, aminopeptidase B (ApsB).ApsBhas 40% sequence identity withApsA, but the encoding genes differ in architecture.apsBis interrupted by elevenintronswhile the ORF ofApsAis interrupted by only one. None of theseintronsshow positional conservation.ApsAandApsBare encoded by similar genes but have different substrate specificities. The results suggest thatapsAandapsBare the result of an ancient duplication event and that both proteins have developed their own niche of intracellular peptide degradation.A thirdaminopeptidase(ApsC) of A.nigerwas cloned using degenerated primers based on sequenced fragments of purifiedApsCfrom an industrial A.nigerstrain. This strain was selected for its high (extracellular)ApsCactivity. The amino acid sequence ofApsCis not similar to any previously characterized aminopeptidase, but shares sequence similarity to mammalianacyl-peptidehydrolases,howeverApsCis not able to hydrolyze N-acetylalanine-pNA, a substrate foracyl- peptidehydrolases.ApsCis active towards N-terminal aromatic amino acids, especially phenylalanine. The pH optimum for this aminopeptidase is between pH 5 and 5.5 which is rather uncommon for acytoplasmicaminopeptidase.The fourthaminopeptidasedescribed in this thesis isPapA.PapAis capable of hydrolyzing N-terminalprolineandhydroxyproline. This enzyme was described earlier inprokaryotes,however,PapAis the first enzyme of this class of enzymes described in eukaryotes.Together these four aminopeptidases are responsible for the breakdown of a whole range of peptide fragments. However, as discussed, there are still some aminopeptidases to be found. In this world were genomic sequences become available fast the actual cloning of a gene is facilitated and less time consuming, however, the biochemical characterization of the aminopeptidases is still very important as well as the study of all the interactions of genes and enzymes in the breakdown of a (large) peptide into its amino acids.