Characterisation of Aspergillus niger prolyl aminopeptidase

Basten, Daniëlle E. J. W.; Moers, Antoine P. H. A.; Ooyen, A.J.J. van; Schaap, Peter J.

doi:10.1007/s00438-004-1094-5

Cited by 29 publications

(25 citation statements)

References 28 publications

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“…PAP have been identified and characterised, e.g. in the ascomycetes A. oryzae and A. niger ( Matsushita-Morita et al 2010, Basten et al 2005. Both PAP have similar properties with a native molecular weight of~315 kDa and seem to be composed of six monomeric units.…”

Section: Fungimentioning

confidence: 98%

Prolyl-specific peptidases for applications in food protein hydrolysis

Mika

Zorn

Rühl

2015

Appl Microbiol Biotechnol

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Various food proteins including, e.g. gluten, collagen and casein are rich in L-proline residues. Due to the cyclic structure of proline, these proteins are well protected from enzymatic degradation by typical digestive enzymes. Proline-specific peptidases (PsP) belong to different families of hydrolases acting on peptide bonds (EC 3.4.x.x). They occur in various organisms including bacteria, fungi, plants and insects. Based on their biochemical characteristics, PsP type enzymes are further grouped into different subclasses of which prolyl aminopeptidases (EC 3.4.11.5, PAP), prolyl carboxypeptidases (EC 3.4.17.16, PCP) and prolyl oligopeptidases/prolyl endopeptidases (EC 3.4.21.26, POP/PEP) are of major interest for applications in food biotechnology. This mini review summarises the biochemical assays employed for these subclasses of PsP and their structural properties and the reaction mechanisms. A special focus was set on PsP derived from fungi and insects and important industrial applications in the field of food biotechnology. The degradation of gluten and collagen as well as the hydrolysis of bitter peptides are discussed.

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Section: Fungimentioning

confidence: 98%

Prolyl-specific peptidases for applications in food protein hydrolysis

Mika

Zorn

Rühl

2015

Appl Microbiol Biotechnol

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“…In contrast, earlier studies of intracellular aminopeptidases from Aspergillus have indicated that expression is constitutive (Basten et al, 2001(Basten et al, , 2003(Basten et al, , 2005). This study definitively shows induction of TePAP when the peptide hydrolysate from casein (a protein rich in proline) is present in growth medium supplemented with a favoured carbon source (glucose).…”

Section: Discussionmentioning

confidence: 85%

“…A number of PAP-type activities in fungi have been reported (Bolumar et al, 2003;Fuke & Matsuoka, 1993;Hiwatashi et al, 2004), but no corresponding protein sequences have been assigned for these enzymes. However, more recently, one study by Basten et al (2005) identified a gene encoding PapA from Aspergillus niger and characterized the recombinant protein.…”

Section: Introductionmentioning

confidence: 99%

Characterization of a multimeric, eukaryotic prolyl aminopeptidase: an inducible and highly specific intracellular peptidase from the non-pathogenic fungus Talaromyces emersonii

et al. 2009

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Fungi are capable of degrading proteins in their environment by secreting peptidases. However, the link between extracellular digestion and intracellular proteolysis has scarcely been investigated. Mycelial lysates of the filamentous fungus Talaromyces emersonii were screened for intracellular peptidase production. Five distinct proteolytic activities with specificity for the p-nitroanilide (pNA) peptides Suc-AAPF-pNA, Suc-AAA-pNA, K-pNA, F-pNA and P-pNA were identified. The native enzyme responsible for the removal of N-terminal proline residues was purified to homogeneity by ammonium sulfate fractionation followed by five successive chromatographic steps. The enzyme, termed Talaromyces emersonii prolyl aminopeptidase (TePAP), displayed a 50-fold specificity for cleaving N-terminal Pro-X (k cat /K m 52.1¾10 6 M "1 s "1 ) compared with Ala-X or Val-X bonds. This intracellular aminopeptidase was optimally active at pH 7.4 and 50 6C. Peptide sequencing facilitated the design of degenerate oligonucleotides from homologous sequences encoding putative fungal proline aminopeptidases, enabling subsequent cloning of the gene. TePAP was shown to be relatively uninhibited by classical serine peptidase inhibitors and to be sensitive to selected cysteine-and histidine-modifying reagents, yet gene sequence analysis identified the protein as a serine peptidase with an a/b hydrolase fold. Northern analysis indicated that Tepap mRNA levels were regulated by the composition of the growth medium. Highest Tepap transcript levels were observed when the fungus was grown in medium containing glucose and the protein hydrolysate casitone. Interestingly, both the induction profile and substrate preference of this enzyme suggest potential co-operativity between extracellular and intracellular proteolysis in this organism. Gel filtration chromatography suggested that the enzyme exists as a 270 kDa homo-hexamer, whereas most bacterial prolyl aminopeptidases (PAPs) are monomers. Phylogenetic analysis of known PAPs revealed two diverse subfamilies that are distinguishable on the basis of primary and secondary structure and appear to correlate with the subunit composition of the native enzymes. Sequence comparisons revealed that PAPs with key conserved topological features are widespread in bacterial and fungal kingdoms, and this study identified many putative PAP candidates within sequenced genomes. This work represents, to our knowledge, the first detailed biochemical and molecular analysis of an inducible PAP from a eukaryote and the first intracellular peptidase isolated from the thermophilic fungus T. emersonii.Abbreviations: AEBSF, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride; DEPC, diethylpyrocarbonate; E-64, trans-epoxy-succinyl-L-leucylamido-(4-guanidino)-butane; NEM, N-ethylmaleimide; PAP, prolyl aminopeptidase; PCMB, p-chloromercuribenzoate; pNA, p-nitroanilide; Pro-AMC, L-proline-7-amino-4-methylcoumarin; TLCK, N-tosyl-L-lysine chloromethyl ketone; TPCK, N-tosyl-L-phenylalanine chloromethyl ketone.3These authors contrib...

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“…The other group consists of a multimeric protein that is either a tetramer or a hexamer obtained from Aeromonas sobria (Kitazono et al. 1994a), A. niger (Basten et al. 2005) and Streptomyces aureofaciens (Uraji et al.…”

Section: Introductionmentioning

confidence: 99%

Characterization of recombinant prolyl aminopeptidase from Aspergillus oryzae

Matsushita-Morita

Furukawa

Suzuki

et al. 2010

Journal of Applied Microbiology

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Aims: Prolyl aminopeptidase (PAP) degrades only amino‐terminal proline from peptides. The food‐grade fungus Aspergillus oryzae produces this enzyme only in small amounts. In this paper, we present efficient production of recombinant PAP with an overexpression system of A. oryzae and characterization of its biochemical properties. Methods and Results: The gene encoding PAP was overexpressed as a His‐tag fusion protein under a taka‐amylase gene (amyB) promoter with a limited expressing condition in A. oryzae. The PAP activity in the mycelia grown in rich medium containing glucose (repressing condition) was twice that in starch (inducing condition). The enzyme prepared as cell‐free extract was partially purified through two‐step column chromatography. The PAP was estimated to be a hexameric protein and exhibited salt tolerance against NaCl of up to 4 mol l−1. Conclusions: Aspergillus oryzae PAP was produced under the repressing condition of amyB promoter in a PAP‐overexpressing strain and purified 1800‐folds. Overproduction of PAP under promoter‐inducing conditions led to an increase in inactive PAP, possibly because of irregular folding. Significance and Impact of the Study: PAP with a high specific activity and salt tolerance may be used effectively in the manufacturing processes of fermented foods.

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Characterisation of Aspergillus niger prolyl aminopeptidase

Cited by 29 publications

References 28 publications

Prolyl-specific peptidases for applications in food protein hydrolysis

Prolyl-specific peptidases for applications in food protein hydrolysis

Characterization of a multimeric, eukaryotic prolyl aminopeptidase: an inducible and highly specific intracellular peptidase from the non-pathogenic fungus Talaromyces emersonii

Characterization of recombinant prolyl aminopeptidase from Aspergillus oryzae

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