A non-specific aminopeptidase from Aspergillus

A fermentation broth supernatant of the Aspergillus oryzae strain ATCC20386 contains aminopeptidase activity that releases a wide variety of amino aci ds from natural peptides. The supernatant was fractionated by anion exchang e chromatography. Based on the primary amino acid sequence data obtained fr om proteins in certain fractions, polymerase chain reaction (PCR) primers w ere made and a PCR product was generated. This PCR product was used to scre en an A. oryzae cDNA library from which the full length gene was then obtai ned. Fusarium venenatum and A. oryzae were used as hosts for gene expressio n. Transformed strains of both F. venenatum and A. oryzae over-expressed an active aminopeptidase (E.C. 3.4.11), named aminopeptidase II. The recombin ant enzyme from both fungal hosts appeared as smears on sodium dodecyl sulf ate-polyacrylamide gel electrophoresis. After deglycosylation of the N-link ed sugars, both samples were a sharp band at similar to 56 kDa and had iden tical N-terminal amino acid sequences. Aminopeptidase II is a metalloenzyme with, presumably, Zn in the active site. Using various natural peptides an d para-nitroanilides (pNAs) of amino acids as substrates, the aminopeptidas e was found to be non-specific. Only X-Pro bonds demonstrated resistance to hydrolysis catalyzed by this aminopeptidase. The optimal enzyme activity w as observed at pH 9.5 and 55 degrees C. Among amino acid pNAs, Leu-pNA appe ars to have the highest value of bimolecular constant of 40 min(-1) mM(-1) (k(cat) = 230 min(-1); K-m = 5.8 mM) at pH 7.5 and 21 degrees C. Among Xaa- Ala-Pro-Tyr-Lys-amide pentapeptides, the velocity of catalytic hydrolysis a t pH 7.5 and 21 degrees C was in a decreasing order: Pro, Ala, Leu, Gly and Glu. (C) 2000 Elsevier Science B.V. All rights reserved.