Purification and Properties of Neutral Proteinase II from Aspergillus oryzae

Nakadai, Tadanobu; Nasuno, Seiichi; Iguchi, Nobuyoshi

doi:10.1271/bbb1961.37.2703

Cited by 43 publications

(17 citation statements)

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“…jhmigatus secretes two other proteinases when the fungus is cultivated at neutral pH in the presence of collagen as the sole nitrogen and carbon source: a serine or alkaline proteinase (ALP) and a metalloproteinase (MEP) which are closely related to the alkaline proteinase and the neutral proteinase I secreted by A. olyzae, respectively [21,22]. However A. oryzae secretes a second metalloproteinase or neutral proteinase II [23], which appears to be absent in A. fumigatus.…”

Section: Resultsmentioning

confidence: 99%

Molecular cloning and sequencing of the gene encoding an extracellular aspartic proteinase fromAspergillus fumigatus

Reichard¹,

Monod

Rüchel³

1995

FEMS Microbiology Letters

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Oligonucleotide primers based on conserved regions of the aspergillopepsins (EC 3.4.23.18) were used to PCR amplify a 650 bp segment of the gene encoding the extracellular aspartic proteinase (PEP) from Aspergillus Fumigatus. The segment was used as a probe for isolating and sequencing the gene from a genomic library of the fungus. Likewise the cDNA was amplified by reverse PCR, cloned and sequenced. The pep gene was found to consist of four exons encoding for 395 aa. The pre-proenzyme deduced has an N-terminal leader sequence of 70 aa preceding the sequence of the mature enzyme consisting of 325 aa with a calculated molecular mass of 34.4 kDa and an isoelectric point of 3.95. The N-terminal sequence of the mature enzyme matched the N-terminal aa sequence of PEP exactly. The nucleotide and the aa sequences of the pre-proenzyme were 70% and 71% homologous to the corresponding sequences of the aspergillopepsin from A. niger var. awamori. Southern analysis of digested genomic A. fumigatus DNA with a specific PCR probe suggested the presence of a single copy of the pep gene.

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Section: Resultsmentioning

confidence: 99%

Molecular cloning and sequencing of the gene encoding an extracellular aspartic proteinase fromAspergillus fumigatus

Reichard¹,

Monod

Rüchel³

1995

FEMS Microbiology Letters

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“…En revanche, par leur spécificité; leur pH optimal d'action et leur poids moléculaire, elles se distinguent nettement des autres métalloprotéases microbiennes, en particulier des métalloprotéases «neutres », telles la thermolysine. Elles appartiennent au sous-groupe des métalloprotéases «acides» auquel s'apparentent également les enzymes synthétisées par Aspergillus sojae (SEKINE, 1972) et A. oryzae (NAKADAI et al, 1973). Cette différence est d'ailleurs confirmée par le fait que le phosphoramidon, un inhibiteur des métalloprotéases neutres, est sur elles sans action.…”

Section: B Les Métalioprotéasesunclassified

Les activités biochimiques des Penicillium utilisés en fromagerie

Cerning

Gripon

Lamberet³

et al. 1987

Lait

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Le P. camemberti et le P. roquejorti constituent respectivement la moisissure superficielle dominante des fromages de type camembert et la moisissure interne des fromages à pâte persillée. Ces deux espèces appartiennent à la section des asymmetrica qui comporte cinq sous-sections dont les lanata (P. camemberti) et les velutina (P. roque-[orti). Toutes les souches ne possèdent pas les mêmes caractéristiques et diverses formes peuvent être reconnues parmi les souches de chacune des deux espèces. Les comportements physiologiques de P. camemberti et de P. roquejorti sont assez différents; la vitesse de croissance du premier est faible, celle du deuxième est plus rapide. La sensibilité au sel du P. camemberti est très faible, le P. roqueforti est stimulé par des faibles teneurs en sel. La capacité des deux espèces à consommer l'acide lactique et les lactates aboutit à la désacidification des pâtes fromagères et entraîne des répercussions majeures en fromagerie. P. camembertiet P. roquejorti possèdent des systèmes protéolytiques présentant entre eux de très grandes analogies et synthétisent tous deux une aspartyl protéase et une métalloprotéase, et des exopeptidases (aminopeptidases et carboxypeptidases). Le niveau de production des diverses enzymes protéolytiques de P, camemberti étudié sur un assez grand nombre de souches varie relativement peu d'une souche à l'autre. L'action in vitro des enzymes protéolytiques sur les caséines met en évidence une analogie d'action entre les enzymes de P. camemberti ct P, roquejorti. Cette analogie est plus marquée pour les aspartyl protéases que pour les métalloprotéases. La protéolyse est qualitativement assez proche dans les camemberts et les fromages à pâte persillée et reflète bien l'homologie observée au niveau des systèmes protéolytiques des deux moisissures. Toutefois l'hydrolyse est plus poussée dans les pâtes persillées.P. roquejorti produit deux lipases, l'une ayant un optimum d'action à pH 6,5 (lipase acide), l'autre à pH 8,0-8,3. P. camemberti ne produit qu'une seule lipase de pH optimal d'action alcalin. Les lipases alcalines des deux espèces possèdent des caractéristiques d'action assez semblables, toutes deux sont plus actives sur la tributyrine que sur les autres triglycérides homogènes et leurs activités relatives sur trioléine sont proches de 20 % de l'activité sur tributyrine. Elles se différencient par leur action sur la tricapryline et la tricaprine avec des vitesses d'hydrolyse relatives de 91 et 62 % pour la lipase de P. roquejorti et de 65 et 27 % pour celle de P. camemberti. Le pH est un facteur important de production de l'une ou l'autre lipase de P. roquejorti, il en va de même pour la lipase alcaline de P. camemberti.L'activité lipasique dans les Summary Biochemical activities 0/ Penicillium used in cheese makingPenicillium camemberti and P. roquejorti represent respectively the main superficial mold of camembert type cheese and the internal mold of blue veined cheese. These species belong to the asymmetrica section which is divided into five sub-sect...

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“…In a manner similar to that of P-I, alkaline proteases of A. oryzae (3, 13, 24), A. sojae (5a), and A. flavus (25) were found to be either inhibited by diisopropyl fluorophosphate or PMSF, and maximum activities fell within the pH range of 8 to 10.5 (3,7,11,13,24). As with P-II, neutral proteases of A. oryzae (12,19,20) and A. flavus (8), were inhibited by EDTA, and the optimum pH values for activities of neutral proteases of A. oryzae (3,19,20) and A. sojae were in the range of 5.5 to 7.0. The temperature for maximum enzyme activity for P-I and P-II (50 to 5500) was also the same as that reported for A. oryzae (3, 12).…”

Section: Reaction Mixtureamentioning

confidence: 81%

“…columnaris, it was cleared as aflatoxin negative (4) before being recommended for routine use in soy sauce factories in Thailand, and it was of interest to observe the result of its extended use in a factory setting. Although there have been a number of studies (7,11,(14)(15)(16)(17)(18)(19)(20)(21)22) on the biology of soy sauce fungi and on the purification and characterization of their proteases, most of these investigations have been carried out on the Japanese soy sauce fungus Aspergillus oryzae. This study was undertaken as part of a program to evaluate the use ofA.…”

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confidence: 99%

Isolation of Alkaline and Neutral Proteases from Aspergillus flavus var. columnaris , a Soy Sauce Koji Mold

Impoolsup

Bhumiratana

Flegel

1981

Appl Environ Microbiol

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Two different extracellular proteases, protease I (P-I), an alkaline protease, and protease II (P-II) a neutral protease, from Aspergillus flavus var. columnaris were partially purified by using (NH4)2SO4 precipitation, diethylaminoethyl-Sephadex A-50 chromatography, carboxymethylcellulose CM-52 chromatography, and Sephadex G-100 gel filtration. The degree of purity was followed using polyacrylamide gel electrophoresis. The activity of P-I was completely inhibited by 0.1 mM phenylmethylsulfonyl fluoride, and that of P-II was completely inhibited by 1 mM ethylenediaminetetraacetate. By using these inhibitors with extracts of wheat bran koji, the proportions of total activity that could be assigned to P-I and P-II were 80 and 20%, respectively. This compared favorably with activities estimated by using polyacrylamide gel electrophoresis slices (82 and 18%, respectively). Extracts from factory-run soybean koji gave comparable results. Both enzymes demonstrated maximum activity at 50 to 55°C and only small changes in activity between pH 6 and 11. For P-I, activity was somewhat higher from pH 8.0 to 11.0, whereas for P-II it was somewhat higher from pH 6 to 9. In the presence of 18% NaCl, the activities of both P-I and P-II dropped by approximately 90 and 85%, respectively. P-I was inferred to possess aminopeptidase activity since it could hydrolyze L-leucyl-p-nitroanilide hydrochloride. P-II was devoid of such activity. The ramifications of the results for factory-produced soy sauce koji are discussed.

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Purification and Properties of Neutral Proteinase II from Aspergillus oryzae

Cited by 43 publications

References 0 publications

Molecular cloning and sequencing of the gene encoding an extracellular aspartic proteinase fromAspergillus fumigatus

Molecular cloning and sequencing of the gene encoding an extracellular aspartic proteinase fromAspergillus fumigatus

Les activités biochimiques des Penicillium utilisés en fromagerie

Isolation of Alkaline and Neutral Proteases from Aspergillus flavus var. columnaris , a Soy Sauce Koji Mold

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