Purification and Properties of Acid Carboxypeptidase III from Aspergillus oryzae

Nakadai, Tadanobu; Nasuno, Seiichi; Iguchi, Nobuyoshi

doi:10.1271/bbb1961.36.1481

Cited by 17 publications

(11 citation statements)

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“…This is consistent with the findings that glutamic acid is the one of the most abundant free amino acids found in matured tofuyo [27,29]. Z-Glu-Tyr was also reported to be the best substrate of acid carboxypeptidases III, IV, O-1, and O-2 from A. oryzae [21,22,23], carboxypeptidases Z-1 and Z-2 from A. zychae [16], and carboxypeptidase S1 from P. janthinellum [9], while Z-Glu-Phe was the best substrate of serine carboxypeptidase from P. carneus [25]. In the present study, both Z-Glu-Tyr and Z-Glu-Phe were good substrates of carboxypeptidase from M. purpureus.…”

Section: Discussionsupporting

confidence: 91%

“…It is considered to serve as a key enzyme in the production of flavorful amino acids during the maturation of tofuyo. Although a large number of carboxypeptidases have been isolated from various species of fungi such as Aspergillus saitoi [10], A. oryzae [17,19,20,21,22,23,24], A. niger [4,14], Absidia zychae [16], Mucor racemsus [7], Penicillium janthinellum [6,9,31], and Pycnoporus sanguineus [12], the Monascus enzyme has not yet been reported. In this study, we describe the purification and characterization of carboxypeptidase produced by M. purpureus IFO 4478.…”

Section: Introductionmentioning

confidence: 99%

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Purification and characterization of a new type of serine carboxypeptidase from Monascus purpureus

Liu

Tachibana

Taira

et al. 2004

Journal of Industrial Microbiology and Biotechnology

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Carboxypeptidase produced by Monascus purpureus IFO 4478 was purified to homogeneity. The purified enzyme is a heterodimer with a molecular mass of 132 kDa and consists of two subunits of 64 and 67 kDa. It is an acidic glycoprotein with an isoelectric point of 3.67 and 17.0% carbohydrate content. The optimum pH and temperature were 4.0 and 40 degrees C, respectively. The enzyme was stable between pH 2.0 and 8.0 at 37 degrees C for 1 h, and up to 50 degrees C at pH 5.0 for 15 min. The enzyme was strongly inhibited by piperastatin A, diisopropylfluoride phosphate (DFP), phenylmethylsulfonylfluoride (PMSF), and chymostatin, suggesting that it is a chymotrypsin-like serine carboxypeptidase. Monascus purpureus carboxypeptidase was also strongly inhibited by p-chloromercuribenzoic acid (PCMB) but not by ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline, indicating that it requires cysteine residue but not metal ions for activity. Benzyloxycarbonyl- l-tyrosyl- l-glutamic acid (Z-Tyr-Glu), among the substrates tested, was the best substrate of the enzyme. The K(m), V(max), K(cat), and K(cat) /K(m) values of the enzyme for Z-Tyr-Glu at pH 4.0 and 37 degrees C were 0.86 mM, 0.917 mM min(-1), 291 s(-1), and 339 mM(-1 )s(-1), respectively.

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Section: Discussionsupporting

confidence: 91%

Section: Introductionmentioning

confidence: 99%

Purification and characterization of a new type of serine carboxypeptidase from Monascus purpureus

Liu

Tachibana

Taira

et al. 2004

Journal of Industrial Microbiology and Biotechnology

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“…The result that both MpiCP-1 and MpiCP-2 exhibited higher activity towards Z-Tyr-Glu, Z-Ala-Glu and Z-Phe-Ala is consistent with findings that glutamic acid and alanine were the two most abundant free amino acids in the matured tofuyo [40,43]. Z-Glu-Tyr was reported to be the best substrate for acid carboxypeptidase III, IV, O-1 and O-2 from A. oryzae [31,32,36], carboxypeptidases Z-1 and Z-2 from A. zychae [25], and carboxypeptidase S1 from P. janthinellum [12], and Z-Glu-Phe was the best substrate for serine carboxypeptidase from P. carneus [37], while Z-Phe-Leu was the best for the carboxypeptidase from Mucor racemsus [8] and carboxypeptidase Y from yeast [11,17]. In the present study, Z-Glu-Tyr and ZPhe-Leu were also found to be good substrates for MpiCP-1 and MpiCP-2, and Z-Glu-Phe also for MpiCP-1, like the substrates of MpuCP [26].…”

Section: Discussionsupporting

confidence: 89%

Purification and characterization of a high molecular mass serine carboxypeptidase from Monascus pilosus

Liu

Tachibana

Taira

et al. 2004

J IND MICROBIOL BIOTECHNOL

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Two serine carboxypeptidases, MpiCP-1 and MpiCP-2, were purified to homogeneity from Monascus pilosus IFO 4480. MpiCP-1 is a homodimer with a native molecular mass of 125 kDa composed of two identical subunits of 61 kDa, while MpiCP-2 is a high mass homooligomer with a native molecular mass of 2,263 kDa composed of about 38 identical subunits of 59 kDa. This is unique among carboxypeptidases and distinguishes MpiCP-2 as the largest known carboxypeptidase. The two purified enzymes were both acidic glycoproteins. MpiCP-1 has an isoelectric point of 3.7 and a carbohydrate content of 11%, while for MpiCP-2 these values were 4.0 and 33%, respectively. The optimum pH and temperature were around 4.0 and 50 degrees C for MpiCP-1, and 3.5 and 50 degrees C for MpiCP-2. MpiCP-1 was stable over a broad range of pH between 2.0 and 8.0 at 37 degrees C for 1 h, and up to 55 degrees C for 15 min at pH 6.0, but MpiCP-2 was stable in a narrow range of pH between 5.5 and 6.5, and up to 50 degrees C for 15 min at pH 6.0. Phenylmethylsulfonylfluoride strongly inhibited MpiCP-1 and completely inhibited MpiCP-2, suggesting that they are both serine carboxypeptidases. Of the substrates tested, benzyloxycarbonyl-L: -tyrosyl-L: -glutamic acid (Z-Tyr-Glu) was the best for both enzymes. The Km, Vmax, Kcat and Kcat/Km values of MpiCP-1 for Z-Tyr-Glu at pH 4.0 and 37 degrees C were 1.33 mM, 1.49 mM min(-1), 723 s(-1) and 545 mM(-1) s(-1), and those of MpiCP-2 at pH 3.5 and 37 degrees C were 1.55 mM, 1.54 mM min(-1), 2,039 s(-1) and 1,318 mM(-1) s(-1), respectively.

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“…This structure may well be shared by other serine carboxypeptidases characterized by molecular weights of approximately 100000 which have not been investigated in similar details (11,14,28,30,34,35). In contrast to this, fungal enzymes like carboxypeptidase Y, penicillocarboxypeptidase S-I and S-2, and carboxypeptidase III from Aspergillus oryzae are monomers with a single peptide chain and molecular weights from 45000 to 65000 (22,24,36) and thus clearly distinct from the carboxypeptidases found in higher plants.…”

Section: Discussionmentioning

confidence: 90%

Isolation of a carboxypeptidase from malted barley by affinity chromatography

Breddam

Sørensen

1983

Carlsberg Res. Commun.

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A carboxypeptidase was isolated from malted barley by affinity chromatography in a yield of approximately 30%. The specific activity was higher than previously obtained for similar enzymes. The enzyme was a dimer where each of the monomers consisted of two peptide chains linked by disulfide bridges. Each monomer contained a single suifhydryl group, which was inaccessible to p-hydroxymercuribenzoate and ELLMAN' S reagent when the enzyme was in its native state. The two peptide chains were separated and the N-terminal sequence of each of them determined. The enzyme contained 6 amino sugar residues and 8% neutral sugar. Isoelectric focusing indicated that the enzyme existed in two forms with pI of 5.65 and 5.73, respectively, but N-terminal sequence determination indicated that they had identical peptide chains. Diisopropyl phosphorofluoridate completely inhibited the enzyme while Hg++ only inhibited the enzyme after deprotonation of an ionizable group on the enzyme with a pK a of approximately 6.7.

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Purification and Properties of Acid Carboxypeptidase III from Aspergillus oryzae

Cited by 17 publications

References 0 publications

Purification and characterization of a new type of serine carboxypeptidase from Monascus purpureus

Purification and characterization of a new type of serine carboxypeptidase from Monascus purpureus

Purification and characterization of a high molecular mass serine carboxypeptidase from Monascus pilosus

Isolation of a carboxypeptidase from malted barley by affinity chromatography

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