Conserved regions within the M1 family of metallo-aminopeptidases have been used to clone a zinc aminopeptidase from the industrially used fungus Aspergillus niger. The derived amino acid sequence of ApsA is highly similar to two yeast zinc aminopeptidases, LAPI and AAPI (53 3 and 50 9 % overall similarity, respectively), two members of the M1 family of metalloaminopeptidases. The encoding gene was successfully overexpressed in A. niger and the overexpressed product was purified and characterized. Aminopeptidase A was found to be active towards a number of amino acid p-nitroanilide (pNA) substrates, viz. K-pNA, R-pNA, L-pNA, M-pNA, A-pNA and F-pNA. The most preferred N-terminal amino acid is lysine and not leucine, arginine or alanine, the N-terminal amino acids preferred by the yeast homologues. The K m and K cat for K-pNA and L-pNA were 0 17 mM and 0 49 µkat mg N1 , and 0 16 mM and 0 31 µkat mg N1 , respectively. The pH optimum of the enzyme is between 7 5 and 8, whereas the enzyme is stable between pH 5 and 8. The enzyme is inhibited by the metal chelators EGTA, EDTA and 1,10-phenanthrolin. Bestatin was also able to inhibit the activity.

We have cloned a gene (papA) that encodes a prolyl aminopeptidase from Aspergillus niger. Homologous genes are present in the genomes of the Eurotiales A. nidulans, A. fumigatus and Talaromyces emersonii, but the gene is not present in the genome of the yeast Saccharomyces cerevisiae. Cell extracts of strains overexpressing the gene under the control of its own promoter showed a fourfold to sixfold increase in prolyl aminopeptidase activity, but no change in phenylalanine or leucine aminopeptidase activity. The overexpressed enzyme was subsequently purified and characterised. The enzyme specifically removes N-terminal proline and hydroxyproline residues from peptides. It is the first enzyme of its kind from a eukaryotic organism that has been characterised.

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A novel enzyme with a specific phenylalanine aminopeptidase activity (ApsC) from Aspergillus niger (CBS 120.49) has been characterized. The derived amino acid sequence is not similar to any previously characterized aminopeptidase sequence but does share similarity with some mammalian acyl-peptide hydrolase sequences. ApsC was found to be most active towards phenylalanine ␤-naphthylamide (F-␤NA) and phenylalanine para-nitroanilide (F-pNA), but it also displayed activity towards other amino acids with aromatic side chains coupled to ␤NA; other amino acids with nonaromatic side chains coupled to either pNA or ␤NA were not hydrolyzed or were poorly hydrolyzed. ApsC was not able to hydrolyze N-acetylalanine-pNA, a substrate for acyl-peptide hydrolases.Many food products contain flavors obtained by the hydrolysis of proteins. These peptides and amino acids can taste sweet, sour, or bitter. Mixtures of endoproteases are often deliberately used in conjunction with exoproteases to improve these food flavors. Exopeptidases can reduce the amount of peptides with undesirable tastes through the removal of a single hydrophobic amino acid, or pairs of them, from the terminal ends. For example, phenylalanine-containing peptides taste Ͼ100-fold more bitter than does free phenylalanine (13,14). Control and termination of the hydrolytic reaction are therefore crucial for obtaining hydrolysates with the desired organoleptic properties.Enzymes from Aspergillus niger have been used in food production for several decades, and five different endoproteases (PepA to PepE [see reference 26 and references therein]), two carboxypeptidases (CpdI and PepF/CpdII [9, 24, 25]), and one aminopeptidase (ApsA [4]) have been cloned and characterized. Experiments have shown that a particular enzyme preparation of A. niger that contains a specific aminopeptidase activity can liberate phenylalanine from proteins that are present in dough and (semi)hard cheeses (8), thereby improving the flavor and aroma of these products. So far, only one phenylalanine-specific aminopeptidase has been characterized, namely, APF1 from the basidiomycetous fungus Schizophyllum commune (6). APF1 is an intracellular zinc metallo-aminopeptidase that can also hydrolyze an N-terminal tyrosine.In this report, we describe a new aminopeptidase. The gene was cloned from A. niger, and purification and characterization of the gene product show that the gene encodes an aminopeptidase that specifically hydrolyzes amino-terminal phenylalanine and other amino acids with aromatic side groups. ). To study the possible regulation of apsC messenger levels by the carbon and nitrogen source, strain N402 was grown on MM supplemented with either 1% glucose, 1% fructose, and 0.6% NH 4 Cl or 0.4% NaNO 3 or on MM supplemented with 1% bovine serum albumin, 1% elastin, or 1% collagen. RNA was isolated from these cultures and subjected to Northern analysis. MATERIALS AND METHODS StrainsPurification of extracellular ApsC from A. niger NRRL 3112 and peptide sequencing. A. niger NRRL 3112 was grown in a...