Kouji Miyauchi

Nihon University, Edo, Tōkyō, Japan

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Publications (11)8.52 Total impact

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    ABSTRACT: Lysozyme (N-acetylmuramide glyconohydrolase, [EC 3.2.1.17]) is one of the best-characterized hydrolases. It cleaves β-1,4 linkages between N-acetyl-D-glucosamine (GlcNAc) homopolymers and GlcNAc-N-acetylmuramic acid heteropolymers, mainly causing lysis in the cell walls of bacteria containing the latter heteropolymers. Lysozymes, which are considered to be self-defense enzymes, are classified into three major types: chicken-type (C-type, GH family 22), goosetype (G-type, GH family 23), and invertebrate-type (I-type, GH family 22). C-type lysozymes have been found in many organisms, including viruses, bacteria, plants, insects, reptiles, birds, and mammals. However, only insufficient information is available relating to shellfish lysozymes. In this study, we attempted to purify and characterize lysozymes from the black-lined limpet Cellana nigrolineata. C. nigrolineata lysozyme (Cn-lysozyme) was purified using ammonium sulfate fractionation (0–70%), gel filtration column chromatography (TOYOPEARL HW-50S), and cation exchange column chromatography (TOYOPEARL CM650-S). The molecular mass of the purified Cn-lysozyme was estimated at 14 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The specific activity of Cn-lysozyme was 43.4 times greater than hen egg white lysozyme. The optimal pH and temperature for Cn-lysozyme activity were 5.0 and 50 °C, respectively. The N-terminal amino acid sequence for Cn-lysozyme showed a 75% similarity to hen egg white lysozyme (C-type). In contrast, it showed only a 7.6% similarity to the sequences of a typical I-type lysozyme, Tapes japonica.
    No preview · Article · Jun 2014
  • Mana Ikeda · Kouji Miyauchi · Masahiro Matsumiya
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    ABSTRACT: A 56 kDa chitinase isozyme (PaChiB) was purified from the stomach of the silver croaker Pennahia argentatus. The optimum pH and pH stability of PaChiB were observed in an acidic pH range. When N-acetylchitooligosaccharides ((GlcNAc)n, n=2 -6) were used as substrates, PaChiB degraded (GlcNAc)4 -6 and produced (GlcNAc)2,3. It degraded (GlcNAc)5 to produce (GlcNAc)2 (23.2%) and (GlcNAc)3 (76.8%). The ability to degrade p-nitrophenyl N-acetylchitooligosaccharides (pNp-(GlcNAc)n, n=2 -4) fell in the following order: pNp-(GlcNAc)3≫ pNp-(GlcNAc)2 pNp-(GlcNAc)4. Based on these results, we concluded that PaChiB is an endo-type chitinolytic enzyme, and that it preferentially hydrolyzes the third glycosidic bond from the non-reducing end of (GlcNAc)n. Activity toward crystalline α- and β-chitin was activated at 124%-185% in the presence of 0.5 M NaCl. PaChiB exhibited markedly high substrate specificity toward crab-shell α-chitin.
    No preview · Article · May 2012 · Bioscience Biotechnology and Biochemistry
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    ABSTRACT: A chitinase was purified from the stomach of a fish, the silver croaker Pennahia argentatus, by ammonium sulfate fractionation and column chromatography using Chitopearl Basic BL-03, CM-Toyopearl 650S, and Butyl-Toyopearl 650S. The molecular mass and isoelectric point were estimated at 42 kDa and 6.7, respectively. The N-terminal amino acid sequence showed a high level of homology with family 18 chitinases. The optimum pH of silver croaker chitinase toward p-nitrophenyl N-acetylchitobioside (pNp-(GlcNAc)2) and colloidal chitin were observed to be pH 2.5 and 4.0, respectively, while chitinase activity increased about 1.5- to 3-fold with the presence of NaCl. N-Acetylchitooligosaccharide ((GlcNAc)n, n = 2–6) hydrolysis products and their anomer formation ratios were analyzed by HPLC using a TSK-GEL Amide-80 column. Since the silver croaker chitinase hydrolyzed (GlcNAc)4–6 and produced (GlcNAc)2–4, it was judged to be an endo-type chitinase. Meanwhile, an increase in β-anomers was recognized in the hydrolysis products, the same as with family 18 chitinases. This enzyme hydrolyzed (GlcNAc)5 to produce (GlcNAc)2 (79.2%) and (GlcNAc)3 (20.8%). Chitinase activity towards various substrates in the order pNp-(GlcNAc)n (n = 2–4) was pNp-(GlcNAc)2 >> pNp-(GlcNAc)4 > pNp-(GlcNAc)3. From these results, silver croaker chitinase was judged to be an enzyme that preferentially hydrolyzes the 2nd glycosidic link from the non-reducing end of (GlcNAc)n. The chitinase also showed wide substrate specificity for degrading α-chitin of shrimp and crab shell and β-chitin of squid pen. This coincides well with the feeding habit of the silver croaker, which feeds mainly on these animals.
    No preview · Article · Jan 2009 · Protein Expression and Purification
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    ABSTRACT: Three seaweed chitinase isozymes (Chi-A, B, and C) were purified from a red algae, Chondrus verrucosus. The molecular weights and isoelectric points were 24.5 kDa and 3.5 for Chi-A, 25.5 kDa and 4.6 for Chi-B, and 24.5 kDa and <3.5 for Chi-C. Optimum pH and temperature were observed at pH 2.0 at 80 degrees C for Chi-A and Chi-C, and at pH 1.0 and 70 degrees C for Chi-B. Toward N-acetylchitooligosaccharide (GlcNAc(n)) (n=2 to 6), Chi-A, B, and C hydrolyzed GlcNAc(5) and GlcNAc(6) and produced GlcNAc(n) (n=2 to 4). GlcNAc(n) (n=3, 4) with the reducing end-side of beta anomer was detected in the hydrolysis products. These results indicate that the reactions of Chi-A, B, and C for GlcNAc(n) were a retaining mechanism similar to that of family 18 chitinase. Toward crystalline chitins, Chi-A, B, and C degraded squid pen beta-chitin more than crab shell or shrimp shell alpha-chitin.
    Full-text · Article · Jan 2009 · Bioscience Biotechnology and Biochemistry
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    ABSTRACT: No abstract is available for this article.
    No preview · Article · Dec 2008 · Fisheries Science

  • No preview · Article · Dec 2007 · Fisheries Science
  • Kouji MIYAUCHI · Masahiro MATSUMIYA · Atsushi MOCHIZUKI
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    ABSTRACT: Lysozyme was purified from purple washington clam Saxidomus purpurata by sequential procedures using Chitopearl Basic BL-01 affinity and TSKgel ODS-120T column chromatographies. Molecular mass of the purified enzyme was estimated to be 12 kDa by SDS-PAGE. Optimum pH of the enzyme was 5.2 toward Micrococcus lysodeikticus cells. The optimum temperature was 50°C. The enzyme was stable in the range of pH 4.8–6.8 and 20–90°C. Further, the N-terminal amino acid sequence of the enzyme showed similarity to lysozymes from invertebrates. However, the specific activity of the enzyme toward M. lysodeikticus cells and p-nitrophenyl penta-N-acetyl-β-chitopentaoside was 143 times and 12 times higher than that of hen egg white lysozyme, respectively.
    No preview · Article · Dec 2006 · Fisheries Science
  • MASAHIRO MATSUMIYA · KOUJI MIYAUCHI · ATSUSHI MOCHIZUKI

    No preview · Article · Apr 2003 · Fisheries Science
  • Kouji Miyauchi · Masahiro Matsumiya · Atsushi Mochizuki

    No preview · Article · Mar 2003 · Nihon-suisan-gakkai-shi
  • Masahiro MATSUMIYA · Kouji MIYAUCHI · Atsushi MOCHIZUKI
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    ABSTRACT: Characterization was investigated on the 38 kDa and 42 kDa chitinase (EC3.2.1.14) isozymes from the liver of Japanese common squid Todarodes pacificus. Optimum pH toward colloidal chitin was observed at pH 3.0 for the 38 kDa chitinase, and pH 3.0 and 9.0 for the 42 kDa chitinase. Km and kcat of the 38 kDa and 42 kDa chitinases toward a longer substrate, glycol chitin, were 0.071 mg/mL and 1.22/s, and 0.074 mg/mL and 0.196/s, respectively. Alternatively, strong substrate inhibition of both chitinases were observed toward a short substrate, N-acetylchitopentaose (GlcNAc5). Both chitinases decomposed not only chitin but also chitosan (D. A. 95%). The cleavage pattern and reaction rate were investigated using N-acetylchitooligosaccharides (GlcNAcn, n = 2–6). Both chitinases hydrolyzed GlcNAcn (n = 4,5, and 6). The release of GlcNAc was not observed. The speed of the reaction was observed to be in the following order: GlcNAc4 > GlcNAc5 > GlcNAc6 for the 38 kDa chitinase, and GlcNAc6 > GlcNAc5 > GlcNAc4 for the 42 kDa chitinase. Both the chitinases released p-nitrophenol from p-nitrophenyl GlcNAcn (n = 2, 3, and 4). N-terminal amino acid sequences of the 38 kDa and 42 kDa chitinases were YLLSXYFTNWSQYRPGAGKYFPQNI and EYRKVXYYTNWSQYREVPAKFFPEN, respectively.
    No preview · Article · Jun 2002 · Fisheries Science
  • Kouji Miyauchi · Masahiro Matsumiya · Atsushi Mochizuki
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    ABSTRACT: Lysozyme was purified from brackishwater clam Corbicula japonica by sequential procedures with ammonium sulfate fractionation, and CHITOPEARL BASIC BL-01 affinity, Hydroxyapatite, S-Sepharose Fast Flow, and CM-TOYOPEARL 650S column chromatographies. The purified enzyme showed a single protein band on polyacrylamide gel electrophoresis (PAGE), and its molecular weight was estimated to be 12,000 by SDS-PAGE. Optimum pH of the enzyme was 4.8 toward Micrococcus lysodeikticus cells. The optimum temperature was 70°C. The enzyme was stable in the range of pH 3.0-6.8 and 20-90°C, respectively. The specific activity of the enzyme toward M. lysodeikticus cells was 256-fold higher than that of hen egg white lysozyme. The N-terminal amino acid sequence and amino acid composition of the enzyme were not similar to those of hen egg white lysozyme, suggesting a genetical difference of both enzymes.
    No preview · Article · Jan 2000 · NIPPON SUISAN GAKKAISHI