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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.
Bioscience Biotechnology and Biochemistry 05/2012; 76(5):971-9. · 1.28 Impact Factor
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Fisheries Science 04/2012; 74(6):1360-1362. · 0.94 Impact Factor
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Fisheries Science 04/2012; 73(6):1404-1406. · 0.94 Impact Factor
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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.
Bioscience Biotechnology and Biochemistry 01/2009; 72(12):3091-9. · 1.28 Impact Factor
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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.
Fisheries Science 12/2006; 72(6):1300 - 1305. · 0.94 Impact Factor
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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.
Fisheries Science 06/2002; 68(3):603 - 609. · 0.94 Impact Factor
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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.
Protein Expression and Purification.
