Purification and some properties of β-N-Acetyl-D-glucosaminidase from viscera of green crab (Scylla serrata)
Abstract β-N-Acetyl-D-glucosaminidase was purified from viscera of green crab (Scylla serrata) by extraction with 0.01 M Tris-HCl buffer (pH 7.5) containing 0.2 M NaCl, ammonium sulfate fractionation, and then chromatography on Sephadex G-100 and DEAE-cellulose (DE-32). The purified enzyme showed a single band on polyacrylamide gel electrophoresis, and the specific activity was determined to be 7990 U/mg. The molecular weight of the whole enzyme was determined to be 132.0 kD, and the enzyme is composed of two identical subunits with molecular mass of 65.8 kD. The optimum pH and optimum temperature of the enzyme for the hydrolysis of p-nitrophenyl-N-acetyl-β-D-glucosaminide (pNP-NAG) were found to be at pH 5.6 and at 50°C, respectively. The study of its stability showed that the enzyme is stable in the pH range from 4.6 to 8.6 and at temperatures below 45°C. The kinetic behavior of the enzyme in the hydrolysis of pNP-NAG followed Michaelis-Menten kinetics with Km of 0.424 ± 0.012 mM and Vmax of 17.65 ± 0.32 µmol/min at pH 5.8 and 37°C, and the activation energy was determined to be 61.32 kJ/mol. The effects of some metal ions on the enzyme were surveyed, and the results show that $ Na^{+} $ and $ K^{+} $ have no effects on the enzyme activity; $ Mg^{2+} $ and $ Ca^{2+} $ slightly activate the enzyme, while $ Ba^{2+} $, $ Zn^{2+} $, $ Mn^{2+} $, $ Hg^{2+} $, $ Pb^{2+} $, $ Cu^{2+} $, and $ Al^{3+} $ inhibit the enzyme to different extents..
| Medienart: |
Artikel |
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| Erscheinungsjahr: |
2006 |
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| Erschienen: |
2006 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:71 |
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| Enthalten in: |
Biochemistry - 71(2006), Suppl 1 vom: Jan., Seite S55-S59 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Zhang, Ji-Ping [VerfasserIn] |
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| Links: |
Volltext [lizenzpflichtig] |
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| Anmerkungen: |
© Pleiades Publishing, Inc. 2006 |
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| doi: |
10.1134/S0006297906130098 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
OLC2050560443 |
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| 100 | 1 | |a Zhang, Ji-Ping |e verfasserin |4 aut | |
| 245 | 1 | 0 | |a Purification and some properties of β-N-Acetyl-D-glucosaminidase from viscera of green crab (Scylla serrata) |
| 264 | 1 | |c 2006 | |
| 336 | |a Text |b txt |2 rdacontent | ||
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| 338 | |a Band |b nc |2 rdacarrier | ||
| 500 | |a © Pleiades Publishing, Inc. 2006 | ||
| 520 | |a Abstract β-N-Acetyl-D-glucosaminidase was purified from viscera of green crab (Scylla serrata) by extraction with 0.01 M Tris-HCl buffer (pH 7.5) containing 0.2 M NaCl, ammonium sulfate fractionation, and then chromatography on Sephadex G-100 and DEAE-cellulose (DE-32). The purified enzyme showed a single band on polyacrylamide gel electrophoresis, and the specific activity was determined to be 7990 U/mg. The molecular weight of the whole enzyme was determined to be 132.0 kD, and the enzyme is composed of two identical subunits with molecular mass of 65.8 kD. The optimum pH and optimum temperature of the enzyme for the hydrolysis of p-nitrophenyl-N-acetyl-β-D-glucosaminide (pNP-NAG) were found to be at pH 5.6 and at 50°C, respectively. The study of its stability showed that the enzyme is stable in the pH range from 4.6 to 8.6 and at temperatures below 45°C. The kinetic behavior of the enzyme in the hydrolysis of pNP-NAG followed Michaelis-Menten kinetics with Km of 0.424 ± 0.012 mM and Vmax of 17.65 ± 0.32 µmol/min at pH 5.8 and 37°C, and the activation energy was determined to be 61.32 kJ/mol. The effects of some metal ions on the enzyme were surveyed, and the results show that $ Na^{+} $ and $ K^{+} $ have no effects on the enzyme activity; $ Mg^{2+} $ and $ Ca^{2+} $ slightly activate the enzyme, while $ Ba^{2+} $, $ Zn^{2+} $, $ Mn^{2+} $, $ Hg^{2+} $, $ Pb^{2+} $, $ Cu^{2+} $, and $ Al^{3+} $ inhibit the enzyme to different extents. | ||
| 700 | 1 | |a Chen, Qing-Xi |4 aut | |
| 700 | 1 | |a Wang, Qin |4 aut | |
| 700 | 1 | |a Xie, Jin-Jin |4 aut | |
| 773 | 0 | 8 | |i Enthalten in |t Biochemistry |d Nauka/Interperiodica, 1936 |g 71(2006), Suppl 1 vom: Jan., Seite S55-S59 |w (DE-627)129065668 |w (DE-600)1109-5 |w (DE-576)014396874 |x 0006-2979 |7 nnns |
| 773 | 1 | 8 | |g volume:71 |g year:2006 |g number:Suppl 1 |g month:01 |g pages:S55-S59 |
| 856 | 4 | 1 | |u https://doi.org/10.1134/S0006297906130098 |z lizenzpflichtig |3 Volltext |
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