A non-specific aminopeptidase from Aspergillus

Blinkovsky, Alexander M.; Byun, Tony; Brown, Kimberly M.; Golightly, Elizabeth J.; Klotz, Alan V.

doi:10.1016/s0167-4838(00)00064-9

Cited by 29 publications

(28 citation statements)

References 19 publications

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“…In the case of industrially used filamentous fungi, like Aspergillus spp., only aminopeptidase activities from Aspergillus oryzae have been described. A. oryzae produces at least seven aminopeptidase activities (Nakadai & Nasuno, 1977) of which four have been purified (Nakadai & Nasuno, 1977 ;Nakadai et al, 1973a, b, c) and one has been cloned (Blinkovsky et al, 2000). Our aim is to characterize the pathways involved in protein catabolism in Aspergillus niger.…”

Section: Abbreviation : Pna P-nitroanilidementioning

confidence: 99%

Lysine aminopeptidase of Aspergillus niger The EMBL accession number for the sequence reported in this paper is AJ292570.

2001

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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.

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Section: Abbreviation : Pna P-nitroanilidementioning

confidence: 99%

Lysine aminopeptidase of Aspergillus niger The EMBL accession number for the sequence reported in this paper is AJ292570.

2001

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“…To date, several serine-type carboxypeptidases from A. oryzae have been purified and characterized. 9,10,13,14) Carboxypeptidase O is one of the enzymes purified from cultured medium of A. oryzae IAM2640. 13) This enzyme showed broad substrate specificity for N-acylpeptides in an acidic pH range.…”

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confidence: 99%

Molecular Cloning ofocpOEncoding Carboxypeptidase O ofAspergillus oryzaeIAM2640

Morita

Kuriyama

Akiyama

et al. 2010

Bioscience, Biotechnology, and Biochemistry

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“…This enzyme apparently discriminates similar amino acids effectively releasing leucine but not isoleucine or valine (Table I); results for alanine para-nitroanilide and ␤-naphthylamide unequivocally show that the structure of the leaving group for a substrate affects both k cat and K m . Taking this into account and expecting that the substrate preference of an aminopeptidase toward derivatives of amino acids, such as para-nitroanilides, and natural peptides can be substantially different (15), the study of the hydrolysis of non-protected peptides catalyzed by the S. capsulata aminopeptidase was warranted. Comparative analysis of the Pro-pNA and Pro-Ala-Pro-Tyr-Lys-amide highlights the misleading role of the para-nitroanilide group.…”

Section: Discussionmentioning

confidence: 99%

“…We assume that EDTA, another strong chelator of transition metals, shows practically no inhibitory effect because of its voluminous structure and polyanionic nature resulting in its inability to penetrate close enough to the zinc of the catalytic site. A neutral pH optimum (18) for the S. capsulata aminopeptidase and a putative zinc binding domain HEXXH (15) in its amino acid sequence are both indications of a zinc metalloenzyme.…”

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confidence: 99%

Aminopeptidase from Sphingomonas capsulata

Byun

Tang

Sloma

et al. 2001

Journal of Biological Chemistry

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A novel aminopeptidase with unique substrate specificity was purified from a culture broth of Sphingomonas capsulata. This is the first reported aminopeptidase to demonstrate broad substrate specificity and yet release glycine and alanine with the highest efficacy. On a series of pentapeptide amides with different N-terminal amino acids, this enzyme efficiently releases glycine, alanine, leucine, proline, and glutamate with the lowest turnover value of 370 min ؊1 for glutamate. At pH 7.5 (pH optimum) and 25°C, the kinetic parameters for alanine para-nitroanilide were found to be k cat ‫؍‬ 7600 min ؊1and K m ‫؍‬ 14 mM. For alanine ␤-naphthylamide, they were k cat ‫؍‬ 860 min ؊1 and K m ‫؍‬ 6.7 mM. Polymerase chain reaction primers were designed based upon obtained internal sequences of the wild type enzyme. The subsequent product was then used to acquire the fulllength gene from an S. capsulata genomic library. An open reading frame encoding a protein of 670 amino acids was obtained. The translated protein has a putative signal peptide that directs the enzyme into the supernatant. A search of the amino acid sequence revealed no significant homology to any known aminopeptidases in the available data bases.The rising interest, both basic and applied, in aminopeptidases (EC 3.4.11) from different sources has led to the discovery of a number of enzymes that differ from each other in cellular location, catalytic mechanism, and substrate specificity (1-3). The majority of bacterial monoaminopeptidases are intracellular or membrane-bound metalloenzymes (1). Based on substrate specificity, bacterial monoaminopeptidases can be divided into two basic categories, specific aminopeptidases, which release only a limited number of amino acids, and those that are able to liberate a relatively broad spectrum of Nterminal amino acid residues (1).Proline and glycine are among the most difficult residues for aminopeptidases to hydrolyze because of their unique structures. Proline is unusual because of its cyclic structure, and glycine is identified by the lack of a side chain. Nature has developed a family of enzymes that recognize proline exclusively (4). A monoaminopeptidase that preferentially releases glycine with high efficiency has not yet been described and thus would be of high interest.In this report we describe a novel extracellular monoaminopeptidase from Sphingomonas capsulata. This enzyme has a clear preference for N-terminal glycine and alanine. Because of this characteristic, this monoaminopeptidase has the potential to significantly enhance the degree of protein hydrolysis (5) when used as a supplement to endoproteases and other exopeptidases. EXPERIMENTAL PROCEDURES MaterialsChemicals used as buffers and reagents were commercial products of at least reagent grade. para-Nitroanilides of L-amino acids and peptide substrates were from Sigma or Bachem. Pentapeptide amides were synthesized at the Core Laboratories (Louisiana State University). S. capsulata strain IFO 12533 was purchased from the Institute for Fermentatio...

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A non-specific aminopeptidase from Aspergillus

Cited by 29 publications

References 19 publications

Lysine aminopeptidase of Aspergillus niger The EMBL accession number for the sequence reported in this paper is AJ292570.

Lysine aminopeptidase of Aspergillus niger The EMBL accession number for the sequence reported in this paper is AJ292570.

Molecular Cloning ofocpOEncoding Carboxypeptidase O ofAspergillus oryzaeIAM2640

Aminopeptidase from Sphingomonas capsulata

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