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Acid Proteinase

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Kenji Takahashi – One of the best experts on this subject based on the ideXlab platform.

  • identification of a glutamic Acid and an aspartic Acid residue essential for catalytic activity of aspergillopepsin ii a non pepsin type Acid Proteinase
    Journal of Biological Chemistry, 2000
    Co-Authors: Xiang-ping Huang, Hideshi Inoue, Naofumi Kagami, Masaki Kojima, Osamu Makabe, Takao Kimura, Koichi Suzuki, Kenji Takahashi

    Abstract:

    Abstract Aspergillopepsin II from Aspergillus niger var. macrosporus is a non-pepsin type or pepstatin-insensitive Acid Proteinase. To identify the catalytic residues of the enzyme, all Acidic residues that are conserved in the homologous Proteinases of family A4 were replaced with Asn, Gln, or Ala using site-directed mutagenesis. The wild-type and mutant pro-enzymes were heterologously expressed in Escherichia coli and refolded in vitro. The wild-type pro-enzyme was shown to be processed into a two-chain active enzyme under Acidic conditions. Most of the recombinant mutant pro-enzymes showed significant activity under Acidic conditions because of autocatalytic activation except for the D123N, D123A, E219Q, and E219A mutants. The D123A, E219Q, and E219A mutants showed neither enzymatic activity nor autoprocessing activity under Acidic conditions. The circular dichroism spectra of the mutant pro- and mature enzymes were essentially the same as those of the wild-type pro- and mature enzyme, respectively, indicating that the mutant pro-enzymes were correctly folded. In addition, two single and one double mutant pro-enzyme, D123E, E219D, and D123E/E219D, did not show enzymatic activity under Acidic conditions. Taken together, Glu-219 and Asp-123 are deduced to be the catalytic residues of aspergillopepsin II.

  • Aspergillus niger Acid Proteinase A
    Advances in experimental medicine and biology, 1998
    Co-Authors: Kenji Takahashi, Naofumi Kagami, Xiang-ping Huang, Masaki Kojima, Hideshi Inoue

    Abstract:

    Aspergillus niger var. macrosporus produces two kinds of extracellular Acid Proteinases, i.e., Proteinase B (aspergillopepsin I or proctase B) and Proteinase A (aspergillopepsin II or proctase A).1 Proteinase B is a typical pepsin-type aspartic Proteinase, whereas Proteinase A is a non-pepsin type Acid Proteinase rather insensitive to specific inhibitors of aspartic Proteinases, including pepstatin, DAN and EPNP.2 Proteinase A has a molecular mass of 22,265 dalton, and is composed of two peptide chains, namely Lchain (39 residues) and Hchain (173 residues), which are noncovalently bound to each other.3,4 It has no similarity in amino Acid sequence with ordinary pepsin-type aspartic Proteinases and appears to have fairly different substrate specificity. The active site residues have not been identified in the previous studies, although the results of chemical modification as well as its pH-activity profile indicated that certain carboxyl groups are involved in the activity.

  • Acid Proteinase from nepenthes distillatoria badura
    Advances in Experimental Medicine and Biology, 1998
    Co-Authors: Senarath B. P. Athauda, Hideshi Inoue, Akihiro Iwamatsu, Kenji Takahashi

    Abstract:

    Plant aspartic Proteinases have so far received much less attention in contrast to the well characterized mammalian, fungal and viral aspartic Proteinases.1 They are widely distributed in the plant kingdom, and have been detected in seeds, leaves and flowers of different plants as well as in the digestive fluid of some insectivorous species.1 Aspartic Proteinases from barley, rice and cardoon flower have been well characterized and their cDNA-derived primary structures have been reported.2–4 However, only a few studies were reported on Proteinases of insectivorous plants.5–8 Insectivorous plant Nepenthes distillatoria is available in Sri Lanka in a large quantity and will be a good source of the insectivorous plant Proteinases. They are interesting not only from the view point of plant physiology, but also from the view point of structure/function relationship and molecular evolution of aspartic Proteinases.

Hideshi Inoue – One of the best experts on this subject based on the ideXlab platform.

  • identification of a glutamic Acid and an aspartic Acid residue essential for catalytic activity of aspergillopepsin ii a non pepsin type Acid Proteinase
    Journal of Biological Chemistry, 2000
    Co-Authors: Xiang-ping Huang, Hideshi Inoue, Naofumi Kagami, Masaki Kojima, Osamu Makabe, Takao Kimura, Koichi Suzuki, Kenji Takahashi

    Abstract:

    Abstract Aspergillopepsin II from Aspergillus niger var. macrosporus is a non-pepsin type or pepstatin-insensitive Acid Proteinase. To identify the catalytic residues of the enzyme, all Acidic residues that are conserved in the homologous Proteinases of family A4 were replaced with Asn, Gln, or Ala using site-directed mutagenesis. The wild-type and mutant pro-enzymes were heterologously expressed in Escherichia coli and refolded in vitro. The wild-type pro-enzyme was shown to be processed into a two-chain active enzyme under Acidic conditions. Most of the recombinant mutant pro-enzymes showed significant activity under Acidic conditions because of autocatalytic activation except for the D123N, D123A, E219Q, and E219A mutants. The D123A, E219Q, and E219A mutants showed neither enzymatic activity nor autoprocessing activity under Acidic conditions. The circular dichroism spectra of the mutant pro- and mature enzymes were essentially the same as those of the wild-type pro- and mature enzyme, respectively, indicating that the mutant pro-enzymes were correctly folded. In addition, two single and one double mutant pro-enzyme, D123E, E219D, and D123E/E219D, did not show enzymatic activity under Acidic conditions. Taken together, Glu-219 and Asp-123 are deduced to be the catalytic residues of aspergillopepsin II.

  • Aspergillus niger Acid Proteinase A
    Advances in experimental medicine and biology, 1998
    Co-Authors: Kenji Takahashi, Naofumi Kagami, Xiang-ping Huang, Masaki Kojima, Hideshi Inoue

    Abstract:

    Aspergillus niger var. macrosporus produces two kinds of extracellular Acid Proteinases, i.e., Proteinase B (aspergillopepsin I or proctase B) and Proteinase A (aspergillopepsin II or proctase A).1 Proteinase B is a typical pepsin-type aspartic Proteinase, whereas Proteinase A is a non-pepsin type Acid Proteinase rather insensitive to specific inhibitors of aspartic Proteinases, including pepstatin, DAN and EPNP.2 Proteinase A has a molecular mass of 22,265 dalton, and is composed of two peptide chains, namely Lchain (39 residues) and Hchain (173 residues), which are noncovalently bound to each other.3,4 It has no similarity in amino Acid sequence with ordinary pepsin-type aspartic Proteinases and appears to have fairly different substrate specificity. The active site residues have not been identified in the previous studies, although the results of chemical modification as well as its pH-activity profile indicated that certain carboxyl groups are involved in the activity.

  • Acid Proteinase from nepenthes distillatoria badura
    Advances in Experimental Medicine and Biology, 1998
    Co-Authors: Senarath B. P. Athauda, Hideshi Inoue, Akihiro Iwamatsu, Kenji Takahashi

    Abstract:

    Plant aspartic Proteinases have so far received much less attention in contrast to the well characterized mammalian, fungal and viral aspartic Proteinases.1 They are widely distributed in the plant kingdom, and have been detected in seeds, leaves and flowers of different plants as well as in the digestive fluid of some insectivorous species.1 Aspartic Proteinases from barley, rice and cardoon flower have been well characterized and their cDNA-derived primary structures have been reported.2–4 However, only a few studies were reported on Proteinases of insectivorous plants.5–8 Insectivorous plant Nepenthes distillatoria is available in Sri Lanka in a large quantity and will be a good source of the insectivorous plant Proteinases. They are interesting not only from the view point of plant physiology, but also from the view point of structure/function relationship and molecular evolution of aspartic Proteinases.

Masaaki Yasuda – One of the best experts on this subject based on the ideXlab platform.

  • application of an Acid Proteinase from monascus purpureus to reduce antigenicity of bovine milk whey protein
    Journal of Industrial Microbiology & Biotechnology, 2011
    Co-Authors: P Nilantha L Lakshman, Shinjiro Tachibana, Hirohide Toyama, Toki Taira, Toshihiko Suganuma, Worapot Suntornsuk, Masaaki Yasuda

    Abstract:

    An Acid Proteinase from Monascus purpureus No. 3403, MpuAP, was previously purified and some characterized in our laboratory (Agric Biol Chem 48:1637–1639, 1984). However, further information about this enzyme is lacking. In this study, we investigated MpuAP’s comprehensive substrate specificity, storage stability, and prospects for reducing antigenicity of whey proteins for application in the food industry. MpuAP hydrolyzed primarily five peptide bonds, Gln4–His5, His10–Leu11, Ala14–Leu15, Gly23–Phe24 and Phe24–Phe25 in the oxidized insulin B-chain. The lyophilized form of the enzyme was well preserved at 30–40°C for 7 days without stabilizers. To investigate the possibility of reducing the antigenicity of the milk whey protein, enzymatic hydrolysates of the whey protein were evaluated by inhibition ELISA. Out of the three main components of whey protein, casein and α-lactalbumin were efficiently degraded by MpuAP. The sequential reaction of MpuAP and trypsin against the whey protein successfully degraded casein, α-lactalbumin and β-lactoglobulin with the highest degree of hydrolysis. As a result, the hydrolysates obtained by using the MpuAP–trypsin combination showed the lowest antigenicity compared with the single application of pepsin, trypsin or pepsin–trypsin combination. Therefore, the overall result suggested that the storage-stable MpuAP and trypsin combination will be a productive approach for making hypoallergic bovine milk whey protein hydrolysates.

  • production of angiotensin i converting enzyme inhibitory peptides from soybean protein with monascus purpureus Acid Proteinase
    Process Biochemistry, 2005
    Co-Authors: Megumi Kuba, C Tana, Shinkichi Tawata, Masaaki Yasuda

    Abstract:

    Abstract Soybean proteins, β-conglycinin and glycinin were hydrolysed by an Acid Proteinase from Monascus purpureus . The degree of hydrolysis and inhibitory activities of angiotensin I-converting enzyme (ACE) increased with increasing proteolysis time. After 10 h of incubation, the IC 50 values of the β-conglycinin and glycinin hydrolysates were determined as 0.126 mg/ml and 0.148 mg/ml, respectively. Four ACE inhibitory peptides were isolated from the soybean protein hydrolysates and identified by protein sequencer. ACE inhibitory peptides isolated from the β-conglycinin hydrolysate were identified as LAIPVNKP (IC 50  = 70 μM) and LPHF (670 μM), and those from the glycinin hydrolysate as SPYP (850 μM) and WL (65 μM). The inhibitory activity of SPYP markedly increased after successive digestion by pepsin, chymotrypsin and trypsin in vitro.

  • degradation of soybean protein by an Acid Proteinase from monascus anka
    Food Science and Technology International Tokyo, 1998
    Co-Authors: Masaaki Yasuda, Maki Sakaguchi

    Abstract:

    Degradation of soybean protein by Monascus-Proteinase was investigated in order to reveal the role of the enzyme in the process of tofuyo ripening. The ratio of trichloroacetic Acid-insoluble nitrogen of soybean protein to the total nitrogen in the reaction mixture decreased with increasing enzymatic reaction time. It was found that the digestion of soybean protein by this enzyme progressed as follows: initially, α’-, α-, and β-subunits in β-conglycinin, and then, the Acidic subunit in glycinin were degraded. However, the basic subunit of glycinin still remained, and some polypeptide bands (around 10 kDa) were formed during the enzyme reaction. The degradation rate of soybean protein by this enzyme was affected by the ethyl alcohol concentration in the reaction mixtures.