Enzymes
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N-Acetylneuraminic Acid Aldolase ( N-Acetylneuraminate Pyruvate Lyase [EC 4.1.3.3] )
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Origin
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: Escherichia coli
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| Reaction |
: N-Acetylneuraminate ↔ N-Acetyl-D-mannosamine + Pyruvate
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Appearance
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: White amorphous powder
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| Activity |
: More than 30 units/mg protein
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| Unit definition |
: One unit is the amount of enzyme required to liberates 1 μmol of N-Acetylmannosamine (or Pyruvic Acid) per minute at pH 7.7 at 37oC, using N-Acetylneuraminic Acid (NANA) as a substrate.
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Storage
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: Stable for one year when stored below 5oC. For prolonged storage, keep at -20oC. |
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Contaminations
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: Free from NADH oxidase |
| Properties 1) 2) | |
| M. W. | … Approx. 98,000 Da (gel filtration) |
| Optimum pH | … 7.5 ~ 8.0 |
| pH stability | … 6.0 ~ 9.0 |
| Thermal stability | … below 65oC (pH 7.0, 20 min) |
| Substrate specificity | … N-Glycolylneuraminic Acid (NGNA) is cleaved as well as NANA. Km = 3.6 mM (NANA), 4.3 mM (NGNA) |
| Use | : Enzymatic determination of Sialic Acid and enzymatic syntheses of novel Sialic Acid derivatives. |
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References
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: 1) Y. Uchida, Y. Tsukada and T. Sugimori, J. Biochem., 96, 507 (1984)
2) Y. Ohta, M. Shimosaka, K. Murata, Y. Tsukada and A. Kimura, Appl. Microbiol. Biotechnol., 24, 386 (1986)
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Neuraminidase (Sialidase) (Acylneuraminyl Hydrolase [EC 3.2.1.18])
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Origin
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: Arthrobacter ureafaciens
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| Reaction |
: Sialyl compound → Sialic Acid + Asialocompound
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Appearance
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: White amorphous powder
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| Activity |
: More than 80 units/mg protein for N-Acetylneuraminyllactose (NANA-lactose)
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| Unit definition |
: One unit is the amount of enzyme required to liberate 1 μmol of N-Acetylneuraminic Acid (NANA) per minute at pH 5.0 at 37oC.
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Storage
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: Stable for one year when stored below 5oC. For prolonged storage, keep at -20oC.
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Contaminations
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: Enzyme activities mentioned below cannot be detected. 1) Protease, N-Acetylneuraminic Acid Aldolase, Glycosidase such as α-Glucosidase, β-Glucosidase, α-Galactosidase, β-Galactosidase, α-Mannosidase, α-Fucosidase, N-Acetyl-α-glucosaminidase, N-Acetyl-β-glucosaminidase, N-Acetyl-αgalactosaminidase, N-Acetyl-β-galactosaminidase, N-Acetyl-α-mannosaminidase and N-Acetyl-β-mannosaminidase. |
| Properties 2) 3) | |
| M. W. | … Approx. 52,000 Da, 66,000 Da and 88,000 Da (gel filtration, SDS-PAGE) |
| Optimum pH | … 4.5 ~ 5.5 (NANA-lactose as a substrate) |
| pH stability | … 4.5 ~ 9.5 |
| Thermal stability | … below 60oC (pH 5.0, 20 min) |
| Substrate specificity | …The a-ketosidic linkage of N-Glycolylneuraminic Acid (NGNA) can be hydrolyzed as well as that of NANA. This enzyme cleaves a(2→3), a(2→6) and a(2→8) linkages of N-Acetylneuraminic Acid in glycoconjugates. The acivity is independent on Ca2+ and is not inhibited by EDTA, which is in striking contrast to Vibrio cholerae Neuraminidase, and is not or slightly inhibited by inhibitors such as Monoiodoacetate, p-Chloromercuribenzoate and HgCl2, which is in striking contrast to Clostridium perfringens Neuraminidase. |
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References
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: 1) Y. Uchida, Y. Tsukada and T. Sugimori, J. Biochem., 82, 1425 (1977)
2) Y. Uchida, Y. Tsukada and T. Sugimori, J. Biochem., 86, 1573 (1979)
3) Y. Ohta, Y. Tsukada and T. Sugimori, J. Biochem., 106, 1086 (1989)
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Neuraminidase Isozyme S
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Origin
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: Arthrobacter ureafaciens
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Appearance
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: White amorphous powder.
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| Activity |
: More than 80 units/mg protein for N-Acetylneuraminyllactose (NANA-lactose).
More than 20 units/mg protein for bovine submaxillary mucin. [at pH 5.0] More than 40 units/mg protein for Colominic Acid. [at pH 4.5] More than 60 units/mg protein for bovine brain ganglioside. [at pH 4.0] |
| Unit definition |
: One unit is the amount of enzyme required to liberate 1 µmol of N-Acetylneuraminic Acid (NANA) per minute at pH 5.0 at 37°C, using N-Acetylneuraminyllactose as a substrate.
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Storage
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: Stable for one year when stored below 5°C. For prolonged storage, keep at -20°C. |
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Contaminations
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: Enzyme activities mentioned below cannot be detected. Protease , N-Acetylneuraminic Acid Aldolase, Glycosidase such as α-Glucosidase, β-Glucosidase, α-Galactosidase, β-Galactosidase, α-Mannosidase, α-Fucosidase, N-Acetyl-α-glucosaminidase, N-Acetyl-β-glucosaminidase, N-Acetyl-α-galactosaminidase, N-Acetyl-β-galactosaminidase, N-Acetyl-α-mannosaminidase and N-Acetyl-β-mannosaminidase. |
| Properties | |
| M. W. | … Approx. 52,000 Da (gel filtration, SDS-PAGE) |
| Optimum pH | … 3.8 ~ 4.4 ( for bovine brain ganglioside ) |
| pH stability | … 3.5 ~ 10.0 |
| Thermal stability | … below 60°C (pH 5.0, 20 min) |
| Substrate specificity | … Isozyme S cleaves α(2 → 3), α(2 → 6) and α(2 → 8) linkages of N-Acetylneuraminic Acid in glycoconjugates. In the absence of detergents and calcium ion, the enzyme hydrolyzes N-Acetylneuraminosyl moiety of polysialogangliosides and produces monosialoganglioside GM1, while in the presence of detergents ( Na-cholate, Triton X-100 etc. ), GM1 is further desialylated to asialoganglioside GA1. the character of the enzyme is similar to Vibrio cholerae Neuraminidase except requirement of Ca2+ in the latter. |
NADH Oxidase
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Origin
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: Bacillus licheniformis
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| Reaction |
: NADH + H+ + O2 ↔ NAD+ + H2O2
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Appearance
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: White amorphous powder
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| Activity |
: More than 50 units/mg protein
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| Unit definition |
: One unit is the amount of enzyme required to oxidize 1 μmol of NADH per minute at pH7.0 at 30oC.
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Storage
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: Stable for one year when stored below 5oC and also stable at room temperature for at least one week. For prolonged storage, keep at -20oC. |
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Contaminations
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: Sometimes, trace amount of catalase might be detected. Therefore, the addition of 10 mM NaN3 into the reaction mixture is recommended when the complete elimination of catalase is needed. |
| Properties | |
| M. W. | … Approx. 240,000 Da |
| Optimum pH | … 6.5 ~ 7.5 |
| Optimum Temperature | … 45oC |
| pH stability | … 7.0 ~ 8.5 |
| Thermal stability | … below 30oC (pH 7.5, 10 min) and below 40oC (in the coexistence of 0.1% bovine serum albumin,pH 7.5,10 min) … 3.2x10-5 M (NADH), 6.7x10-6 M (FAD) |
| Substrate specificity | … In the absence of added FAD both NADH and NADPH are oxidized equally, but by the addition of FAD (about 30 μM) to reaction mixture the reaction velocity to NADH is accelerated about 20 ~ 30 times in contrast to 2 ~ 3 times of NADPH. Accordingly, the substrate specificity of NADH is about 10 times larger than that of NADPH in the presence of added FAD. |
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