The Purification and Characterization of Prolyl Aminopeptidase from Penicillium camemberti

Fuke, Yoko; Matsuoka, Hiroatsu

doi:10.3168/jds.s0022-0302(93)77582-7

Cited by 22 publications

(20 citation statements)

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“…The estimated molecular mass (370 kDa) of the native enzyme of D. hansenii is close to those (400 to 270 kDa) determined for enzymes of eukaryotes (7,14,21,35). The enzymes from prokaryotes have lower molecular mass, varying between 150 and 30 kDa (8).…”

Section: Discussionmentioning

confidence: 81%

“…The optimum temperature of the majority of PAPs is between 37 and 55°C (17,32). The PAP of Penicillium camemberti has optimal activity at 45°C, as is the case for the PAP of D. hansenii (7).…”

Section: Discussionmentioning

confidence: 99%

“…Most of the purified PAPs have optimal pHs of between 7 and 8 (7,14,16,32,35). The optimum temperature of the majority of PAPs is between 37 and 55°C (17,32).…”

Section: Discussionmentioning

confidence: 99%

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Purification and Characterization of a Prolyl Aminopeptidase from Debaryomyces hansenii

Bolumar

Sanz

Aristoy

et al. 2003

Appl Environ Microbiol

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A prolyl aminopeptidase (PAP) (EC 3.4.11.5) was isolated from the cell extract of Debaryomyces hansenii CECT12487. The enzyme was purified by selective fractionation with protamine and ammonium sulfate, followed by two chromatography steps, which included gel filtration and anion-exchange chromatography. The PAP was purified 248-fold, with a recovery yield of 1.4%. The enzyme was active in a broad pH range (from 5 to 9.5), with pH and temperature optima at 7.5 and 45°C. The molecular mass was estimated to be around 370 kDa. The presence of inhibitors of serine and aspartic proteases, bestatin, puromycin, reducing agents, chelating agents, and different cations did not have any effect on the enzyme activity. Only iodoacetate, p-chloromercuribenzoic acid, and Hg 2؉ , which are inhibitors of cysteine proteases, markedly reduced the enzyme activity. The K m for proline-7-amido-4-methylcoumarin was 40 M. The enzyme exclusively hydrolyzed N-terminal-proline-containing substrates. This is the first report on the identification and purification of this type of aminopeptidase in yeast, which may contribute to the scarce knowledge about D. hansenii proteases and their possible roles in meat fermentation.

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Section: Discussionmentioning

confidence: 81%

Section: Discussionmentioning

confidence: 99%

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Purification and Characterization of a Prolyl Aminopeptidase from Debaryomyces hansenii

Bolumar

Sanz

Aristoy

et al. 2003

Appl Environ Microbiol

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“…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%

“…Proline, the product of PAP, has multiple physiological functions in micro-organisms, plants and animals, and its accumulation has been implicated in stress responses in yeast and in the suppression of apoptosis in fungi (Takagi, 2008). PAP is an important enzyme industrially, as fungi that possess intracellular proline aminopeptidase-type activity have been extensively used in flavour development of food products (Bolumar et al, 2003;Fuke & Matsuoka, 1993).…”

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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Extremophiles for Hydrolytic Enzymes Productions: Biodiversity and Potential Biotechnological Applications

Kour

Rana

Kaur

et al. 2019

Bioprocessing for Biomolecules Production

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The Purification and Characterization of Prolyl Aminopeptidase from Penicillium camemberti

Cited by 22 publications

References 22 publications

Purification and Characterization of a Prolyl Aminopeptidase from Debaryomyces hansenii

Purification and Characterization of a Prolyl Aminopeptidase from Debaryomyces hansenii

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

Extremophiles for Hydrolytic Enzymes Productions: Biodiversity and Potential Biotechnological Applications

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